C HeLa cells were infected with x - light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy . IPTG was added 30 min prior to fixation , and then samples were labelled with antibody for p62 . The scale bar represents 5 μm . Graph represents mean % ± SEM of Shigella responding to IPTG without ( - ) or with ( + ) p62 from at least 4 independent experiments . Student ' s t - test , * * * = p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C HeLa cells were infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for p62. The scale bar represents 5 μm. Graph represents mean % ± SEM of Shigella responding to IPTG without (-) or with (+) p62 from at least 4 independent experiments. Student's t-test, *** = p<0.001.


D Control ( CTRL ) or isopropanol - treated x - light Shigella GFP were induced with IPTG for 30 min , then labelled with SYTOX Orange for 10 min . The scale bar represents 5 μm .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Control (CTRL) or isopropanol-treated x-light Shigella GFP were induced with IPTG for 30 min, then labelled with SYTOX Orange for 10 min. The scale bar represents 5 μm.


E HeLa cells were infected for 1 h 30 min , treated with ethanol ( CTRL ) or erythromycin ( EM ) for 2 h prior to fixation , and then labelled with antibody for SEPT7 . Graph represents mean % ± SEM of Shigella in SEPT7 cages from 3 independent experiments . Student ' s t - test , * * * = p < 0 . 001 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E HeLa cells were infected for 1 h 30 min, treated with ethanol (CTRL) or erythromycin (EM) for 2 h prior to fixation, and then labelled with antibody for SEPT7. Graph represents mean % ± SEM of Shigella in SEPT7 cages from 3 independent experiments. Student's t-test, *** = p<0.001.


B HeLa cells stably expressing SEPT6 - GFP were infected with S . flexneri AfaI for 4 h , harvested , and then tested with Co - IP . Empty vector and / or uninfected HeLa cells were used in parallel as control . GFP - trap magnetic agarose beads were used to isolate SEPT6 - GFP from cells , and extracts were immunoblotted for SEPT6 , GFP , SEPT7 , or p62 . The lysates were immunoblotted for GAPDH as control for cellular protein levels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B HeLa cells stably expressing SEPT6-GFP were infected with S. flexneri AfaI for 4 h, harvested, and then tested with Co-IP. Empty vector and/or uninfected HeLa cells were used in parallel as control. GFP-trap magnetic agarose beads were used to isolate SEPT6-GFP from cells, and extracts were immunoblotted for SEPT6, GFP, SEPT7, or p62. The lysates were immunoblotted for GAPDH as control for cellular protein levels.


A HeLa cells were labelled with MitoTracker Red CMXRos , treated with cytochalasin D for 30 min , then fixed and labelled for endogenous SEPT7 for confocal microscopy . The scale bar represents 0 . 5 μm . See also Fig EV3A .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A HeLa cells were labelled with MitoTracker Red CMXRos, treated with cytochalasin D for 30 min, then fixed and labelled for endogenous SEPT7 for confocal microscopy. The scale bar represents 0.5 μm. See also Fig EV3A.


B HeLa cells were infected with S . flexneri for 4 h 40 min , labelled with MitoTracker Red CMXRos , fixed and labelled with antibody for endogenous SEPT7 for quantitative confocal microscopy . The scale bar represents 1 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B HeLa cells were infected with S. flexneri for 4 h 40 min, labelled with MitoTracker Red CMXRos, fixed and labelled with antibody for endogenous SEPT7 for quantitative confocal microscopy. The scale bar represents 1 μm.


C HeLa cells stably expressing SEPT6 - GFP were transfected with Mito - BFP for 24 h , infected with Shigella - mCherry for 4 h 40 min , and processed for CLEM . SEPT6 is shown in green , mitochondria in red , and Shigella - mCherry in blue . Septin cages identified by fluorescent light microscopy ( FLM ) were processed for TEM . The right - most image is enlarged from the boxed region in the TEM image , and shows the bacterium ( Sf ) surrounded by a double membrane of autophagy ( Au ) and the mitochondrial membrane ( Mt ) . The scale bar represents 1 μm . See also Fig EV3B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C HeLa cells stably expressing SEPT6-GFP were transfected with Mito-BFP for 24 h, infected with Shigella-mCherry for 4 h 40 min, and processed for CLEM. SEPT6 is shown in green, mitochondria in red, and Shigella-mCherry in blue. Septin cages identified by fluorescent light microscopy (FLM) were processed for TEM. The right-most image is enlarged from the boxed region in the TEM image, and shows the bacterium (Sf) surrounded by a double membrane of autophagy (Au) and the mitochondrial membrane (Mt). The scale bar represents 1 μm. See also Fig EV3B.


D HeLa cells stably expressing SEPT6 - GFP were transfected with Mito - BFP for 24 h , then infected with Shigella - mCherry for 2 h for live confocal microscopy . Time frame sequence shows a septin cage assembling around Shigella . Mitochondria ( circle ) support sites of septin ring assembly around the bacterium . Each frame was acquired every 2 min . The scale bar represents 1 μm . See also Movie EV1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D HeLa cells stably expressing SEPT6-GFP were transfected with Mito-BFP for 24 h, then infected with Shigella-mCherry for 2 h for live confocal microscopy. Time frame sequence shows a septin cage assembling around Shigella. Mitochondria (circle) support sites of septin ring assembly around the bacterium. Each frame was acquired every 2 min. The scale bar represents 1 μm. See also Movie EV1.


E HeLa cells were treated with control ( CTRL ) , Drp1 , or Mfn1 siRNA for 72 h . Whole cell lysates were immunoblotted for Drp1 or Mfn1 to show the efficiency of depletion . GAPDH was used as loading control . siRNA - treated cells were infected with S . flexneri for 4 h 40 min , then fixed for microscopy and stained for endogenous SEPT7 for quantitative confocal microscopy . Graph represents the mean % ± SEM of Shigella inside SEPT7 cages from at least 4 independent experiments per treatment . Student ' s t - test , * * = p < 0 . 01 ; * * * = p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E HeLa cells were treated with control (CTRL), Drp1, or Mfn1 siRNA for 72 h. Whole cell lysates were immunoblotted for Drp1 or Mfn1 to show the efficiency of depletion. GAPDH was used as loading control. siRNA-treated cells were infected with S. flexneri for 4 h 40 min, then fixed for microscopy and stained for endogenous SEPT7 for quantitative confocal microscopy. Graph represents the mean % ± SEM of Shigella inside SEPT7 cages from at least 4 independent experiments per treatment. Student's t-test, ** = p <0.01; *** = p<0.001.


A HeLa cells expressing SEPT6 - GFP were stained with MitoTracker Red CMXRos , and fixed for confocal microscopy . Inset image highlights location of septins at sites of mitochondrial fission . The scale bar represents 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A HeLa cells expressing SEPT6-GFP were stained with MitoTracker Red CMXRos, and fixed for confocal microscopy. Inset image highlights location of septins at sites of mitochondrial fission. The scale bar represents 5 μm.


B HeLa cells were fixed and labelled with antibody against SEPT7 and secondary antibody coupled to HRP , and processed for TEM . The right image is enlarged from the boxed region in the TEM image , and shows a SEPT7 ( S7 ) filament ( labelled in black ) intersecting with the mitochondrial ( Mt ) fission site . The scale bar represents 100 nm . See also Fig EV4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B HeLa cells were fixed and labelled with antibody against SEPT7 and secondary antibody coupled to HRP, and processed for TEM. The right image is enlarged from the boxed region in the TEM image, and shows a SEPT7 (S7) filament (labelled in black) intersecting with the mitochondrial (Mt) fission site. The scale bar represents 100 nm. See also Fig EV4.


C HeLa cells expressing SEPT6 - GFP were stained with MitoTracker Red CMXRos for live SOFI - based super resolution microscopy . Each image was reconstructed from 100 raw TIRF frames using a custom made SOFI - based algorithm . Arrows highlight SEPT6 association with location of mitochondrial fission . The scale bar represents 1 µm . See also Movie EV3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C HeLa cells expressing SEPT6-GFP were stained with MitoTracker Red CMXRos for live SOFI-based super resolution microscopy. Each image was reconstructed from 100 raw TIRF frames using a custom made SOFI-based algorithm. Arrows highlight SEPT6 association with location of mitochondrial fission. The scale bar represents 1 µm. See also Movie EV3.


D HeLa cells expressing SEPT6 - GFP were labelled with MitoTracker Red CMXRos , fixed for widefield immunofluorescence microscopy , and labelled with antibodies to Drp1 . Arrows highlight one example of Drp1 and SEPT6 association with location of mitochondrial fission . The scale bar represents 1 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D HeLa cells expressing SEPT6-GFP were labelled with MitoTracker Red CMXRos, fixed for widefield immunofluorescence microscopy, and labelled with antibodies to Drp1. Arrows highlight one example of Drp1 and SEPT6 association with location of mitochondrial fission. The scale bar represents 1 μm.


E HeLa cells were transfected with Mito - BFP and ActA - SEPT7 - mCherry , fixed and labelled with antibody to Drp1 for widefield immunofluorescence microscopy . The scale bar represents 5 μm . Inset images highlight Drp1 and SEPT7 associated with sites of mitochondrial fission . Pearson ' s correlation coefficient from 3 independent experiments ( mean ± SEM ) shows that mitochondria in ActA - SEPT7 transfected cells recruit significantly more Drp1 than control cells ( Mito - BFP transfected alone ) . Student ' s t - test , * = p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E HeLa cells were transfected with Mito-BFP and ActA-SEPT7-mCherry, fixed and labelled with antibody to Drp1 for widefield immunofluorescence microscopy. The scale bar represents 5 μm. Inset images highlight Drp1 and SEPT7 associated with sites of mitochondrial fission. Pearson's correlation coefficient from 3 independent experiments (mean ± SEM) shows that mitochondria in ActA-SEPT7 transfected cells recruit significantly more Drp1 than control cells (Mito-BFP transfected alone). Student's t-test, * = p<0.05.


A HeLa cells were treated with control ( CTRL ) , SEPT7 , or Drp1 siRNA for 72 h . Whole cell lysate of siRNA - treated cells were immunoblotted for GAPDH , SEPT7 , or Drp1 to show the efficiency of siRNA depletion . GAPDH was used as loading control . siRNA - treated cells were labelled with MitoTracker Red CMXRos , and fixed for confocal microscopy . The scale bar represents 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A HeLa cells were treated with control (CTRL), SEPT7, or Drp1 siRNA for 72 h. Whole cell lysate of siRNA-treated cells were immunoblotted for GAPDH, SEPT7, or Drp1 to show the efficiency of siRNA depletion. GAPDH was used as loading control. siRNA-treated cells were labelled with MitoTracker Red CMXRos, and fixed for confocal microscopy. The scale bar represents 5 μm.


B HeLa cells were treated with control ( CTRL ) , SEPT2 , SEPT7 , SEPT9 , Drp1 , or SEPT7 + Drp1 siRNA for 72 h , labelled with MitoTracker Red CMXRos , and fixed for quantitative confocal microscopy . Graph represents the distribution in length ( μm ; whiskers from min to max ) of mitochondria in siRNA - treated cells from 3 independent experiments ( analysis of at least 100 measurements per biological replicate ) . Student ' s t - test , * * * = p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B HeLa cells were treated with control (CTRL), SEPT2, SEPT7, SEPT9, Drp1, or SEPT7+ Drp1 siRNA for 72 h, labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Graph represents the distribution in length (μm; whiskers from min to max) of mitochondria in siRNA-treated cells from 3 independent experiments (analysis of at least 100 measurements per biological replicate). Student's t-test, *** = p<0.001.


C HeLa cells stably expressing SEPT6 - GFP were harvested , and then tested with Co - IP . HeLa cells expressing GFP were used in parallel as control . GFP - trap magnetic agarose beads were used to isolate SEPT6 - GFP from cells , and extracts were immunoblotted for Drp1 , SEPT2 , SEPT7 , or GFP . The lysates were immunoblotted for GAPDH as control for cellular protein levels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C HeLa cells stably expressing SEPT6-GFP were harvested, and then tested with Co-IP. HeLa cells expressing GFP were used in parallel as control. GFP-trap magnetic agarose beads were used to isolate SEPT6-GFP from cells, and extracts were immunoblotted for Drp1, SEPT2, SEPT7, or GFP. The lysates were immunoblotted for GAPDH as control for cellular protein levels.


D HeLa cells were treated with control ( CTRL ) or SEPT7 siRNA for 72 h , and whole cell lysates were immunoblotted for SEPT7 , Drp1 , phosphorylated Drp1 ( P - Drp1 ) , or Mfn1 . GAPDH was used as loading control . Graph represents the mean % ± SEM of the relative amount of Drp1 , P - Drp1 , Mfn1 , or SEPT7 proteins quantified by densitometry from 4 independent experiments . Student ' s t - test , ns = non - significant ; * * = p < 0 . 01 , * * * = p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, and whole cell lysates were immunoblotted for SEPT7, Drp1, phosphorylated Drp1 (P-Drp1), or Mfn1. GAPDH was used as loading control. Graph represents the mean % ± SEM of the relative amount of Drp1, P-Drp1, Mfn1, or SEPT7 proteins quantified by densitometry from 4 independent experiments. Student's t-test, ns = non-significant; ** = p<0.01, *** = p<0.001.


E HeLa cells were treated with control ( CTRL ) or SEPT7 siRNA for 72 h , labelled with MitoTracker Red CMXRos , then fixed and labelled for endogenous P - Drp1 for quantitative confocal microscopy . Representative images shown here . The scale bar represents 1 μm . The recruitment of P - Drp1 to mitochondria was significantly decreased as compared to control cells as measured by Pearson ' s correlation coefficient from at least 5 independent experiments ( mean ± SEM , analysis of at least 250 cells per biological replicate ) . Student ' s t - test , * = p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, labelled with MitoTracker Red CMXRos, then fixed and labelled for endogenous P-Drp1 for quantitative confocal microscopy. Representative images shown here. The scale bar represents 1 μm. The recruitment of P-Drp1 to mitochondria was significantly decreased as compared to control cells as measured by Pearson's correlation coefficient from at least 5 independent experiments (mean ± SEM, analysis of at least 250 cells per biological replicate). Student's t-test, * = p<0.05.


F HeLa cells were treated with control ( CTRL ) or SEPT7 siRNA for 72 h , labelled with MitoTracker Red CMXRos , then fixed and labelled for endogenous Mfn1 for quantitative confocal microscopy . Representative images shown here . The scale bar represents 1 μm . The recruitment of Mfn1 to mitochondria was significantly increased as compared to control cells as measured by Pearson ' s correlation coefficient from at least 5 independent experiments ( mean ± SEM , analysis of at least 250 cells per biological replicate ) . Student ' s t - test , * = p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, labelled with MitoTracker Red CMXRos, then fixed and labelled for endogenous Mfn1 for quantitative confocal microscopy. Representative images shown here. The scale bar represents 1 μm. The recruitment of Mfn1 to mitochondria was significantly increased as compared to control cells as measured by Pearson's correlation coefficient from at least 5 independent experiments (mean ± SEM, analysis of at least 250 cells per biological replicate). Student's t-test, * = p<0.05.


A HeLa cells stably expressing SEPT6 - GFP were treated with Drp1 siRNA for 72 h , transfected with mito - BFP 24 h , and then infected with S . flexneri - mCherry for 2 h for live confocal microscopy . Representative image shows interplay between two Shigella - septin cages and fused mitochondria . The scale bar represents 1 μm . See also Movie EV3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A HeLa cells stably expressing SEPT6-GFP were treated with Drp1 siRNA for 72 h, transfected with mito-BFP 24 h, and then infected with S. flexneri-mCherry for 2 h for live confocal microscopy. Representative image shows interplay between two Shigella-septin cages and fused mitochondria. The scale bar represents 1 μm. See also Movie EV3.


B HeLa cells were treated with Drp1 siRNA for 72 h , and then infected with x - light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy . IPTG was added 30 min prior to fixation , and then samples were labelled with antibody for SEPT7 . Graph represents mean % ± SEM of Shigella responding to IPTG outside ( - ) or inside ( + ) SEPT7 cages from 3 independent experiments . Student ' s t - test , * * * = p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B HeLa cells were treated with Drp1 siRNA for 72 h, and then infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for SEPT7. Graph represents mean % ± SEM of Shigella responding to IPTG outside (-) or inside (+) SEPT7 cages from 3 independent experiments. Student's t-test, *** = p<0.001.


C HeLa cells were infected with S . flexneri M90T for 4 h 40 min , labelled with MitoTracker Red CMXRos , and fixed for quantitative confocal microscopy . Boxplots show the length of mitochondrial ( μm ; whiskers from min to max ) surrounding Shigella outside ( - ) or inside ( + ) SEPT7 cages from 3 independent experiments ( analysis of at least 130 measurements per biological replicate ) . Student ' s t - test ; * * * = p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C HeLa cells were infected with S. flexneri M90T for 4 h 40 min, labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Boxplots show the length of mitochondrial (μm; whiskers from min to max) surrounding Shigella outside (-) or inside (+) SEPT7 cages from 3 independent experiments (analysis of at least 130 measurements per biological replicate). Student's t-test; *** = p<0.001.


D HeLa cells were either kept uninfected as a control or infected with S . flexneri M90T or S . flexneri ΔIcsA for 4 h 40 min . Samples were labelled with MitoTracker Red CMXRos , and fixed for quantitative confocal microscopy . Boxplots show length of mitochondria ( μm ; whiskers from min to max ) in uninfected cells ( CTRL ) , surrounding S . flexneri M90T ( + M90T ) or S . flexneri ΔIcsA ( + ΔIcsA ) from 3 independent experiments ( analysis of at least 250 measurements per biological replicate ) . Student ' s t - test ; * * * = p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D HeLa cells were either kept uninfected as a control or infected with S. flexneri M90T or S. flexneri ΔIcsA for 4 h 40 min. Samples were labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Boxplots show length of mitochondria (μm; whiskers from min to max) in uninfected cells (CTRL), surrounding S. flexneri M90T (+M90T) or S. flexneri ΔIcsA (+ΔIcsA) from 3 independent experiments (analysis of at least 250 measurements per biological replicate). Student's t-test; *** = p<0.001.


LC - MS / MS measurements of m6A levels in total RNA ( B ) or in poly ( A ) + RNA ( C ) upon KD of predicted methyltransferases in Drosophila S2R + cells . m6A abundance in total RNA is significantly reduced when Mettl5 is depleted , while its depletion has no effect on m6A level in mRNA . As expected , the KD of Mettl3 and Mettl14 reduce m6A levels in mRNA . Bar chart represents the mean ± standard deviation of three technical measurements from three biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( two - tailed t - test ) .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LC-MS/MS measurements of m6A levels in total RNA (B) or in poly(A)+ RNA (C) upon KD of predicted methyltransferases in Drosophila S2R+ cells. m6A abundance in total RNA is significantly reduced when Mettl5 is depleted, while its depletion has no effect on m6A level in mRNA. As expected, the KD of Mettl3 and Mettl14 reduce m6A levels in mRNA. Bar chart represents the mean ± standard deviation of three technical measurements from three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed t-test).


Subcellular localization of Mettl5 - FLAG ( purple ) and Trmt112 - eGFP ( green ) expressed under the control of their own promoter in S2R + cells . 63X magnified merge of immunofluorescently - labelled S2R + cells . Scale bar : 4 . 47 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Subcellular localization of Mettl5-FLAG (purple) and Trmt112-eGFP (green) expressed under the control of their own promoter in S2R+ cells. 63X magnified merge of immunofluorescently-labelled S2R+ cells. Scale bar: 4.47 μm.


Developmental expression of Mettl5 transcript assayed by RT - qPCR analysis . The figure shows mean ± standard deviation of three technical measurements from three biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Developmental expression of Mettl5 transcript assayed by RT-qPCR analysis. The figure shows mean ± standard deviation of three technical measurements from three biological replicates.


In situ hybridization of Mettl5 transcript at different embryonic stages . The central nervous system ( CNS ) is highlighted in the schematics . Data retrieved from FlyExpress 7 . ( http : / / www . flyexpress . net / search . php ? type = image & search = FBgn0036856 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

In situ hybridization of Mettl5 transcript at different embryonic stages. The central nervous system (CNS) is highlighted in the schematics. Data retrieved from FlyExpress 7. (http://www.flyexpress.net/search.php?type=image&search=FBgn0036856).


LC - MS / MS measurements of m6A levels in total RNA of WT and Mettl5 mutant flies . Bars represent mean ± standard deviation of measurements of three biological replicates . * * * P < 0 . 001 ( two - tailed t - test ) .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LC-MS/MS measurements of m6A levels in total RNA of WT and Mettl5 mutant flies. Bars represent mean ± standard deviation of measurements of three biological replicates. ***P<0.001 (two-tailed t-test).


Purified 18S rRNA analyzed for its m6A content by quantitative HPLC . The 18S rRNA was extracted from 40S subunits isolated on sucrose gradients . The calibration control is a commercial source of m6A ( in grey ) . m6A elutes at 48 min .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O

Purified 18S rRNA analyzed for its m6A content by quantitative HPLC. The 18S rRNA was extracted from 40S subunits isolated on sucrose gradients. The calibration control is a commercial source of m6A (in grey). m6A elutes at 48 min.


Mature rRNA analysis on ethidium - stained denaturing agarose gels . The same amounts of total RNA extracted from the indicated flies , and from S2R + cells depleted or not of Mettl5 , were loaded . The 28S / 18S ratio was established by densitometry .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mature rRNA analysis on ethidium-stained denaturing agarose gels. The same amounts of total RNA extracted from the indicated flies, and from S2R+ cells depleted or not of Mettl5, were loaded. The 28S/18S ratio was established by densitometry.


Analysis of 18S rRNA maturation in WT and mutant flies ( Mettl5fs ) . Total RNA extracted from the indicated animals was resolved on denaturing agarose gels and processed for northern blotting with specific probes ( complementary to ITS1 or ITS2 sequences ) . The pre - rRNAs detected accumulate to normal levels indicating that processing is unaffected .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analysis of 18S rRNA maturation in WT and mutant flies (Mettl5fs). Total RNA extracted from the indicated animals was resolved on denaturing agarose gels and processed for northern blotting with specific probes (complementary to ITS1 or ITS2 sequences). The pre-rRNAs detected accumulate to normal levels indicating that processing is unaffected.


Western blot validation of the co - immunoprecipitated proteins Mettl5 - FLAG - Myc and Trmt112 , bearing either a N - terminal HA tag ( top ) or a C - terminal GFP tag ( bottom ) , in S2R + cells . The arrows point at the indicated proteins . HA - Trmt12 migrates below the antibody light chains ( top ) . Trmt112 - eGFP migrates above the heavy chains indicated by an asterisk ( bottom ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot validation of the co-immunoprecipitated proteins Mettl5-FLAG-Myc and Trmt112, bearing either a N-terminal HA tag (top) or a C-terminal GFP tag (bottom), in S2R+ cells. The arrows point at the indicated proteins. HA-Trmt12 migrates below the antibody light chains (top). Trmt112-eGFP migrates above the heavy chains indicated by an asterisk (bottom).


Trmt112 depletion reduces m6A levels in total RNA from Drosophila S2R + cells . Mean ± standard deviation of three technical measurements from three biological replicates . * P < 0 . 05 ( two - tailed t - test ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Trmt112 depletion reduces m6A levels in total RNA from Drosophila S2R+ cells. Mean ± standard deviation of three technical measurements from three biological replicates. *P < 0.05 (two-tailed t-test).


Representative trajectories of WT and Mettl5 mutant flies analysed by the Buridan ' s Paradigm . The blue lines indicate when the fly stops and changes direction . Orientation evaluated with the help of the Buridan ' s paradigm for Mettl5 mutant flies . Orientation was measured as the angular deviation from the straight path needed to move from one landmark to another in the arena . Number of flies tested per genotype : 30 . Mean ± standard deviation . Shapiro - Wilk test was used to test for normal distribution in each group . Normally distributed groups were tested by t - test . Due to multiple comparison Bonferroni correction was applied . ( p < 0 . 05 = * ; p < 0 . 0001 = * * * * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative trajectories of WT and Mettl5 mutant flies analysed by the Buridan's Paradigm. The blue lines indicate when the fly stops and changes direction. Orientation evaluated with the help of the Buridan's paradigm for Mettl5 mutant flies. Orientation was measured as the angular deviation from the straight path needed to move from one landmark to another in the arena. Number of flies tested per genotype: 30. Mean ± standard deviation. Shapiro-Wilk test was used to test for normal distribution in each group. Normally distributed groups were tested by t-test. Due to multiple comparison Bonferroni correction was applied. (p<0.05 = *; p<0.0001=****).


( E - F ) Serum levels of T ( E ) and LH ( F ) in WT males , ARLmon / Y and AR - / Y mice at the age of 13 weeks .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(E-F) Serum levels of T (E) and LH (F) in WT males, ARLmon/Y and AR-/Y mice at the age of 13 weeks.


( D ) RT - qPCR analyses of Rhox5 and Insl3 of testes from 13 - week old WT males , ARLmon / Y and AR - / Y . Expression levels are normalized to WT .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) RT-qPCR analyses of Rhox5 and Insl3 of testes from 13-week old WT males, ARLmon/Y and AR-/Y. Expression levels are normalized to WT.


( A ) Expression levels of genes involved in steroidogenesis normalized to WT . The bar graphs show means ± SEM ( data derived from RNA - seq analysis of testes , biological replicates , n = 5 , one - way ANOVA with Tukey ' s multiple comparisons test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant ) .

(A) Expression levels of genes involved in steroidogenesis normalized to WT. The bar graphs show means ± SEM (data derived from RNA-seq analysis of testes, biological replicates, n = 5, one-way ANOVA with Tukey's multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant).


( B ) Serum levels of A - dione in WT males , ARLmon / Y and AR - / Y mice at the age of 13 weeks .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(B) Serum levels of A-dione in WT males, ARLmon/Y and AR-/Y mice at the age of 13 weeks.


( D ) Reporter gene assays in Hela cells co - transfected with the mouse Insl3 promotor together with the WT or mutant mouse AR .

O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Reporter gene assays in Hela cells co-transfected with the mouse Insl3 promotor together with the WT or mutant mouse AR.


mRNA expression levels extracted from RNA - seq data for Fkbp5 ( D ) in kidneys used for RNA - seq . Expression levels are normalized to WT ORX .

mRNA expression levels extracted from RNA-seq data for Fkbp5 (D) in kidneys used for RNA-seq. Expression levels are normalized to WT ORX.


mRNA expression levels extracted from RNA - seq data for , Odc1 ( E ) and Kap ( F ) in kidneys used for RNA - seq . Expression levels are normalized to WT ORX .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

mRNA expression levels extracted from RNA-seq data for , Odc1 (E) and Kap (F) in kidneys used for RNA-seq. Expression levels are normalized to WT ORX.


( B ) Specific ligand binding assay in COS cells with overload of cold ligand and increasing concentrations of radioactive labeled DHT . Values are normalized to protein expression .

(B) Specific ligand binding assay in COS cells with overload of cold ligand and increasing concentrations of radioactive labeled DHT. Values are normalized to protein expression.


( C ) Western blot on cytoplasmic , nuclear and whole cell extracts derived from Hela cells transfected with human WT or W752R AR followed by stimulation with increasing concentrations of DHT . Both panels represent the same blot . The blot was cut in two parts and antibodies against HSP90 and Lamin A / C were used to confirm cellular fractionation of cytoplasmic and nuclear proteins . For AR visualization ( upper panel ) , longer exposure time was used . # = residual AR expression ( lower panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Western blot on cytoplasmic, nuclear and whole cell extracts derived from Hela cells transfected with human WT or W752R AR followed by stimulation with increasing concentrations of DHT. Both panels represent the same blot. The blot was cut in two parts and antibodies against HSP90 and Lamin A/C were used to confirm cellular fractionation of cytoplasmic and nuclear proteins. For AR visualization (upper panel), longer exposure time was used. # = residual AR expression (lower panel).


( B ) AR occupancy ( blue ) and levels of the histone marks H3K27ac ( green ) and H3K27me3 ( red ) at ARBS within the regulatory regions of Fkbp5 , Kap ( both androgen - regulated in kidney ) and Tox3 ( androgen - regulated in prostate but not in kidney and hence used as a negative control ) were assessed by ChIP - qPCR in kidneys from 13 - week - old ARLmon / Y mice and WT males .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) AR occupancy (blue) and levels of the histone marks H3K27ac (green) and H3K27me3 (red) at ARBS within the regulatory regions of Fkbp5, Kap (both androgen-regulated in kidney) and Tox3 (androgen-regulated in prostate but not in kidney and hence used as a negative control) were assessed by ChIP-qPCR in kidneys from 13-week-old ARLmon/Y mice and WT males.


( C ) Representative pictures of immunofluorescence AR staining ( green ) in kidneys of castrated WT males and ARLmon / Y mice supplemented with vehicle or T . Nuclei are shown in blue .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative pictures of immunofluorescence AR staining (green) in kidneys of castrated WT males and ARLmon/Y mice supplemented with vehicle or T. Nuclei are shown in blue.


( A ) Double hybrid assay in COS cells transfected with WT LBD , W752R LBD or empty vector ( negative control ) in the presence of the LxxLL fragment of SRC1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Double hybrid assay in COS cells transfected with WT LBD, W752R LBD or empty vector (negative control) in the presence of the LxxLL fragment of SRC1.


( B ) Confocal fluorescence microscopy images of AR - WT - BirA * and AR W752R - BirA * expressing HEK293 cells treated in the presence of 50 µM biotin with 100 nM DHT or vehicle ( ethanol ) and with or without 0 . 03 µg / ml TET as indicated . AR - BirA * s were detected with anti - AR ( red ) and biotinylated proteins with fluorescently labeled streptavidin ( green ) . Nuclei were visualized using DAPI .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Confocal fluorescence microscopy images of AR-WT-BirA* and AR W752R-BirA*expressing HEK293 cells treated in the presence of 50 µM biotin with 100 nM DHT or vehicle (ethanol) and with or without 0.03 µg/ml TET as indicated. AR-BirA*s were detected with anti-AR (red) and biotinylated proteins with fluorescently labeled streptavidin (green). Nuclei were visualized using DAPI.


A . STAT3 immunohistochemistry - staining of low STAT3 and high STAT3 PCa samples . Red arrows indicate transformed PCa glands , black arrows indicate pre - transformed normal prostate glands . Scale bar = 100µm . # = sample - IDs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. STAT3 immunohistochemistry- staining of low STAT3 and high STAT3 PCa samples. Red arrows indicate transformed PCa glands, black arrows indicate pre-transformed normal prostate glands. Scale bar = 100µm. # = sample-IDs.


A . Differentially expressed proteins in PtenStat3pc - / - vs . Ptenpc - / - proteomic samples . Colors indicate adj . p - value and log2 - FC . ( Black = Log2 - FC ≤ 1 & adj . p - value ≥ 0 . 05 , orange = Log2 - FC > 1 & adj . p - value ≥ 0 . 05 , red = Log2 - FC > 1 & adj . p - value < 0 . 05 ) . FC = fold change .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Differentially expressed proteins in PtenStat3pc-/- vs. Ptenpc-/- proteomic samples. Colors indicate adj.p-value and log2-FC. (Black = Log2-FC ≤ 1 & adj. p-value ≥ 0.05, orange = Log2-FC > 1 & adj. p-value ≥ 0.05, red = Log2-FC > 1 & adj. p-value < 0.05). FC = fold change.


B . STAT3 immunohistochemistry - staining of wild type , Ptenpc - / - , PtenStat3pc - / - mouse prostates . Scale bar = 100µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. STAT3 immunohistochemistry- staining of wild type, Ptenpc-/-, PtenStat3pc-/- mouse prostates. Scale bar = 100µm.


E . Metabolite concentrations in nmol / µg of 5 metabolites in WT , Ptenpc - / - and PtenStat3pc - / - prostates . Box - plot shows median , 1st and 3rd quartiles , and whiskers extend to ± 1 . 5 interquartile range . Jitter represents biological replicates . ANOVA tests and Tukey multiple comparisons were applied . Red = PtenStat3pc - / - ( n = 3 ) , blue = Ptenpc - / - ( n = 5 ) , grey = wild type ( WT , n = 5 ) , q = adj . p . - value , n . s . = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Metabolite concentrations in nmol/µg of 5 metabolites in WT, Ptenpc-/- and PtenStat3pc-/- prostates. Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents biological replicates. ANOVA tests and Tukey multiple comparisons were applied. Red = PtenStat3pc-/- (n = 3), blue = Ptenpc-/- (n = 5), grey = wild type (WT, n=5), q = adj. p.-value, n.s. = not significant.


A . SDHB protein expression levels in a TMA ( n = 83 ) detected by immunohistochemistry ( IHC ) . Box - plot shows median , 1st and 3rd quartiles , and whiskers extend to ± 1 . 5 interquartile range . Jitter represents single values in groups . Kruskal - Wallis test and Dunn ' s all pairs test were applied . Q = adj . p . - value , n . s . = not significant . TMA = Tissue Micro Array .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. SDHB protein expression levels in a TMA (n = 83) detected by immunohistochemistry (IHC). Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents single values in groups. Kruskal-Wallis test and Dunn's all pairs test were applied. Q = adj. p.-value, n.s. = not significant. TMA = Tissue Micro Array.


B . Representative IHC - staining of SDHB in normal prostate glands and Gleason grade ( GL ) 3 - 5 PCa glands . Scale bar = 100µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Representative IHC- staining of SDHB in normal prostate glands and Gleason grade (GL) 3 - 5 PCa glands. Scale bar = 100µm.


C . IDH2 protein expression levels in a TMA ( n = 83 ) detected by IHC . Box - plot shows median , 1st and 3rd quartiles , and whiskers extend to ± 1 . 5 interquartile range . Jitter represents single values in groups . Kruskal - Wallis test and Dunn ' s all pairs test were applied . Q = adj . p . - value , n . s . = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. IDH2 protein expression levels in a TMA (n = 83) detected by IHC. Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents single values in groups. Kruskal-Wallis test and Dunn's all pairs test were applied. Q = adj. p.-value, n.s. = not significant.


D . Representative IHC staining of IDH2 in normal prostate glands and GL 3 - 5 PCa glands . Scale bar = 100µm .

O O O O O O O O O O O O O O O O O O O O O O O O

D. Representative IHC staining of IDH2 in normal prostate glands and GL 3 - 5 PCa glands. Scale bar = 100µm.


E . Nuclear ( N ) and cytoplasmatic ( C ) STAT3 protein expression levels in a TMA ( n = 83 ) detected by IHC . Box - plot shows median , 1st and 3rd quartiles , and whiskers extend to ± 1 . 5 interquartile range . Jitter represents single values in groups . Kruskal - Wallis test and Dunn ' s all pairs test were applied . Q = adj . p . - value , n . s . = not significant .

E. Nuclear (N) and cytoplasmatic (C) STAT3 protein expression levels in a TMA (n = 83) detected by IHC. Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents single values in groups. Kruskal-Wallis test and Dunn's all pairs test were applied. Q = adj. p.-value, n.s. = not significant.


B . Genes of the Hallmark gene set " Oxidative phosphorylation " that are differentially expressed in low STAT3 vs . high STAT3 tumors . Dotted lines show Log2 - FC = ± 1 ( x - axis ) and adj . p - value = - log10 ( 0 . 05 ) ( y - axis ) . Orange = up - regulated genes , green = down - regulated genes . DE = differentially expressed .

B. Genes of the Hallmark gene set "Oxidative phosphorylation" that are differentially expressed in low STAT3 vs. high STAT3 tumors. Dotted lines show Log2-FC = ±1 (x-axis) and adj. p-value = -log10(0.05) (y-axis). Orange = up-regulated genes, green = down-regulated genes. DE = differentially expressed.


C . - D . Kaplan - Meier plots showing time to biochemical recurrence in months for PDK4 in primary tumors ( C ) and in primary and metastatic tumors combined ( D ) in the MSKCC PCa GSE21032 data set . Groups were generated by a median split . P - values were estimated by Log - rank test ( C - D ) and adjusted with Benjamini - Hochberg method ( D ) . + = censored .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C.- D. Kaplan-Meier plots showing time to biochemical recurrence in months for PDK4 in primary tumors (C) and in primary and metastatic tumors combined (D) in the MSKCC PCa GSE21032 data set. Groups were generated by a median split. P-values were estimated by Log-rank test (C-D) and adjusted with Benjamini-Hochberg method (D). + = censored.


A . STAT3 and PDK4 stratified subgroups were generated by median splits in MSKCC PCa GSE21032 data set . Pearson correlation between STAT3 and PDK4 is shown . Kaplan - Meier plot shows stratified subgroups . P - values were estimated by Log - rank test and adjusted with Benjamini - Hochberg method . Hi = high , lo = low .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. STAT3 and PDK4 stratified subgroups were generated by median splits in MSKCC PCa GSE21032 data set. Pearson correlation between STAT3 and PDK4 is shown. Kaplan-Meier plot shows stratified subgroups. P-values were estimated by Log-rank test and adjusted with Benjamini-Hochberg method. Hi = high, lo = low.


B . Western blot of STAT3 , PDK4 and β - TUBULIN proteins in 22Rv1 cells with or without knockdown of STAT3 . Ctrl = scrambled control , shSTAT3 = short hairpin knockdown of STAT3 .

B. Western blot of STAT3, PDK4 and β-TUBULIN proteins in 22Rv1 cells with or without knockdown of STAT3. Ctrl = scrambled control, shSTAT3 = short hairpin knockdown of STAT3.


C . ChIP assay from IL - 6 stimulated or non - stimulated 22Rv1 cells with or without knockdown of STAT3 was immunoprecipitated with a STAT3 specific antibody ( blue shades ) and IgG antibody as negative control ( orange shades ) followed by qPCR with a promoter - specific primer pair for the PDK4 gene . Bars represent mean ± SD from 2 technical replicates . Precipitated DNA is presented as % of input . One representative experiment is shown . Result of qPCR using primer pair 2 is shown . Ctrl = scrambled control , shSTAT3 = short hairpin knockdown of STAT3 , + IL - 6 = IL - 6 stimulated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. ChIP assay from IL-6 stimulated or non-stimulated 22Rv1 cells with or without knockdown of STAT3 was immunoprecipitated with a STAT3 specific antibody (blue shades) and IgG antibody as negative control (orange shades) followed by qPCR with a promoter- specific primer pair for the PDK4 gene. Bars represent mean ± SD from 2 technical replicates. Precipitated DNA is presented as % of input. One representative experiment is shown. Result of qPCR using primer pair 2 is shown. Ctrl = scrambled control, shSTAT3 = short hairpin knockdown of STAT3, +IL-6 = IL-6 stimulated.


D . Correlation of STAT3 , c - MYC and HIF - 1α with PDK1 - 4 , PDC - genes ( PDHA1 , PDHB , PDHX , DLAT , DLD ) and TCA / OXPHOS genes ( CS , IDH2 , IDH3A , SDHB , SDHC , ATP5A1 , NDUFS1 ) in MSKCC PCa ( GSE21032 ) . Dot colors represent Pearson correlation ( 1 = red , - 1 = blue ) , dot sizes represent adj . p - values ≤ 0 . 05 . Only significant correlations are shown . P - values were adjusted with Benjamini - Hochberg method .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Correlation of STAT3, c-MYC and HIF-1α with PDK1-4, PDC- genes (PDHA1, PDHB, PDHX, DLAT, DLD) and TCA/OXPHOS genes (CS, IDH2, IDH3A, SDHB, SDHC, ATP5A1, NDUFS1) in MSKCC PCa (GSE21032). Dot colors represent Pearson correlation (1 = red, -1 = blue), dot sizes represent adj. p-values ≤ 0.05. Only significant correlations are shown. P-values were adjusted with Benjamini-Hochberg method.


( a ) Fluorescence microscopy of GFP - LC3 - transduced HeLa cells left unstimulated ( Unstim ) or stimulated for 4 h with C12 - iEDAP ( 2 . 5 μg / ml ) or for 2 h with rapamycin ( Rapa ; 50 μg / ml ) . Green , GFP - LC3 ; blue , DAPI ( DNA - intercalating dye ) . Scale bar , 5 μm . ( b ) Quantification of dot - or ring - shaped GFP - LC3 signals ( representing autophagosomes ) in HeLa cells stimulated as described in a .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Fluorescence microscopy of GFP-LC3-transduced HeLa cells left unstimulated (Unstim) or stimulated for 4 h with C12-iEDAP (2.5 μg/ml) or for 2 h with rapamycin (Rapa; 50 μg/ml). Green, GFP-LC3; blue, DAPI (DNA-intercalating dye). Scale bar, 5 μm. (b) Quantification of dot- or ring-shaped GFP-LC3 signals (representing autophagosomes) in HeLa cells stimulated as described in a.


( c ) Confocal microscopy of wild - type and Nod2 - deficient BMDMs transduced with GFP - LC3 ( green ) and left unstimulated or stimulated for 4 h with LPS ( 1 μg / ml ) or MDP ( 100 μg / ml ) or for 2 h with rapamycin ( 50 μg / ml ) . Scale bar , 15 μm . ( d ) Quantification of dot - or ring - shaped GFP - LC3 ( green ) in BMDMs stimulated as described in c .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Confocal microscopy of wild-type and Nod2-deficient BMDMs transduced with GFP-LC3 (green) and left unstimulated or stimulated for 4 h with LPS (1 μg/ml) or MDP (100 μg/ml) or for 2 h with rapamycin (50 μg/ml). Scale bar, 15 μm. (d) Quantification of dot- or ring-shaped GFP-LC3 (green) in BMDMs stimulated as described in c.


( e ) Immunofluorescence microscopy of macrophages recovered from the peritoneal cavities of thioglycolate - injected wild - type , Nod1 - and Nod2 - deficient mice 4 h after intraperitoneal injection of MDP ( 300 μg / ml ) , FK565 ( 300 μg / ml ) or rapamycin ( 300 μg / ml ) ; after centrifugation by cytospin , cells were stained for endogenous LC3 ( red ) and with DAPI ( blue ) . Scale bar , 10 μm . ( f ) Quantification of dot - or ring - shaped LC3 signals in macrophages recovered from the peritoneal cavities of mice treated as described in c . Data are representative of four ( a - d ) or two ( e , f ) independent experiments with at least 100 cells per condition ( error bars ( b , d , f ) , s . d . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) Immunofluorescence microscopy of macrophages recovered from the peritoneal cavities of thioglycolate-injected wild-type, Nod1- and Nod2-deficient mice 4 h after intraperitoneal injection of MDP (300 μg/ml), FK565 (300 μg/ml) or rapamycin (300 μg/ml); after centrifugation by cytospin, cells were stained for endogenous LC3 (red) and with DAPI (blue). Scale bar, 10 μm. (f) Quantification of dot- or ring-shaped LC3 signals in macrophages recovered from the peritoneal cavities of mice treated as described in c. Data are representative of four (a-d) or two (e,f) independent experiments with at least 100 cells per condition (error bars (b,d,f), s.d.).


( a ) Confocal microscopy of wild - type and Nod1 - deficient GFP - LC3 - expressing MEFs infected for 2 h with wild - type S . flexneri M90T - RFP . ( b ) Quantification of the colocalization of GFP - LC3 signals with bacteria in a .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Confocal microscopy of wild-type and Nod1-deficient GFP-LC3-expressing MEFs infected for 2 h with wild-type S. flexneri M90T-RFP. (b) Quantification of the colocalization of GFP-LC3 signals with bacteria in a.


( c ) Confocal microscopy of wild - type and Nod1 - deficient GFP - LC3 - expressing MEFs infected for 2 h with S . flexneri ΔIcsB - RFP . ( d ) Quantification of the colocalization of GFP - LC3 signals with bacteria in c .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Confocal microscopy of wild-type and Nod1-deficient GFP-LC3-expressing MEFs infected for 2 h with S. flexneri ΔIcsB-RFP. (d) Quantification of the colocalization of GFP-LC3 signals with bacteria in c.


( e , f ) Plate assay of bacteria in wild - type or Nod1 - deficient MEFs infected for 0 . 5 h with S . flexneri ΔIcsB in the presence or absence of the autophagy blocker LY294002 ( LY ) or protease inhibitors ( PI ) , followed by 5 . 5 h of incubation in the presence of gentamicin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

(e,f) Plate assay of bacteria in wild-type or Nod1-deficient MEFs infected for 0.5 h with S. flexneri ΔIcsB in the presence or absence of the autophagy blocker LY294002 (LY) or protease inhibitors (PI), followed by 5.5 h of incubation in the presence of gentamicin.


( g - j ) Confocal microscopy ( g , i ) and quantification of the colocalization of GFP - LC3 signals with M90T and ΔIcsB ( h , j ) in wild - type and RIP2 - deficient ( Ripk2 − / − ) GFP - LC3 - expressing MEFs infected for 2 h with wild - type S . flexneri ( M90T ; g , h ) or ΔIcsB S . flexneri ( i , j ) .

(g-j) Confocal microscopy (g,i) and quantification of the colocalization of GFP-LC3 signals with M90T and ΔIcsB (h,j) in wild-type and RIP2-deficient (Ripk2−/−) GFP-LC3-expressing MEFs infected for 2 h with wild-type S. flexneri (M90T; g,h) or ΔIcsB S. flexneri (i,j).


( k ) Enzyme - linked immunosorbent assay of KC secretion by wild - type and RIP2 - deficient MEFs infected for 0 . 5 h with M90T and incubated for 5 . 5 h in the presence of gentamicin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(k) Enzyme-linked immunosorbent assay of KC secretion by wild-type and RIP2-deficient MEFs infected for 0.5 h with M90T and incubated for 5.5 h in the presence of gentamicin.


( l , m ) Confocal microscopy ( l ) and quantification of the colocalization of GFP - LC3 signals ( green ) with M90T and ΔIcsB ( m ) of wild - type and NEMO - deficient ( Ikbkg − / − ) GFP - LC3 - expressing MEFs infected for 0 . 5 h with wild - type S . flexneri ( M90T ) or S . flexneri ΔIcsB and incubated for 1 . 5 h in the presence of gentamicin . Red , RFP . Arrows indicate colocalization of GFP - LC3 signals with S . flexneri . Scale bars , 10 μm . * P 0 . 05 , and * * P 0 . 01 ( t - test ) . Data represent three independent experiments with at least 100 cells each ( error bars , s . e . m . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(l,m) Confocal microscopy (l) and quantification of the colocalization of GFP-LC3 signals (green) with M90T and ΔIcsB (m) of wild-type and NEMO-deficient (Ikbkg−/−) GFP-LC3-expressing MEFs infected for 0.5 h with wild-type S. flexneri (M90T) or S. flexneri ΔIcsB and incubated for 1.5 h in the presence of gentamicin. Red, RFP. Arrows indicate colocalization of GFP-LC3 signals with S. flexneri. Scale bars, 10 μm. *P 0.05, and **P 0.01 (t-test). Data represent three independent experiments with at least 100 cells each (error bars, s.e.m.).


( a ) Immunofluorescence of HeLa cells expressing hemagglutinin - tagged Nod1 ( HA - Nod1 ( red ) ; top ) or HA - Nod2 ( red ; bottom ) . Blue , DAPI .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Immunofluorescence of HeLa cells expressing hemagglutinin-tagged Nod1 (HA-Nod1 (red); top) or HA-Nod2 (red; bottom). Blue, DAPI.


( b ) Immunofluorescence of HeLa cells expressing Myc - tagged FL - ATG16L1 ( Myc - FL - ATG16L1 ) .

O O O O O O O O O O O O O O O O O O O O O O

(b) Immunofluorescence of HeLa cells expressing Myc-tagged FL-ATG16L1 (Myc-FL-ATG16L1).


( c , d ) Immunofluorescence of HeLa cells coexpressing HA - Nod1 ( red ; c ) or HA - Nod2 ( red ; d ) and Myc - FL - ATG16L1 ( green ) . Blue , DAPI .

(c,d) Immunofluorescence of HeLa cells coexpressing HA-Nod1 (red; c) or HA-Nod2 (red; d) and Myc-FL-ATG16L1 (green). Blue, DAPI.


( e ) Immunofluorescence of HeLa cells expressing a Flag - tagged truncated form of ATG16L1 ( Flag - ΔN85 - ATG16L1 ; green ) . Blue , DAPI .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) Immunofluorescence of HeLa cells expressing a Flag-tagged truncated form of ATG16L1 (Flag-ΔN85-ATG16L1; green). Blue, DAPI.


( f ) Immunofluorescence of HeLa cells coexpressing HA - Nod2 ( red ) and Flag - ΔN85 - ATG16L1 ( green ) . Blue , DAPI .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) Immunofluorescence of HeLa cells coexpressing HA-Nod2 (red) and Flag-ΔN85-ATG16L1 (green). Blue, DAPI.


( g ) Immunofluorescence microscopy of Mode - K cells stained for endogenous Nod2 ( green ) and ATG16L1 ( red ) and with DAPI ( blue ) . For a - g , arrows indicate membrane colocalization of ATG16 and Nod2 ; area outlined at left is enlarged at right ( a , b , e ) or in other images ( c , d , f , ) ; scale bars , 8 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g) Immunofluorescence microscopy of Mode-K cells stained for endogenous Nod2 (green) and ATG16L1 (red) and with DAPI (blue). For a-g, arrows indicate membrane colocalization of ATG16 and Nod2; area outlined at left is enlarged at right (a,b,e) or in other images (c,d,f,); scale bars, 8 μm.


( h , i ) Immunoprecipitation ( IP ) and immunoblot ( IB ) analysis of HEK293 cells transfected with Flag - ΔN85 - ATG16L1 ( h ) or Myc - FL - ATG16L1 ( i ) , together with HA - Nod1 , HA - Nod2 or HA - tagged enhanced GFP ( EGFP - HA ) . Ig , immunoglobulin ( loading control ) . Results are representative of one of at least three independent experiments ( a - g ) or one of three independent experiments ( h , i ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h,i) Immunoprecipitation (IP) and immunoblot (IB) analysis of HEK293 cells transfected with Flag-ΔN85-ATG16L1 (h) or Myc-FL-ATG16L1 (i), together with HA-Nod1, HA-Nod2 or HA-tagged enhanced GFP (EGFP-HA). Ig, immunoglobulin (loading control). Results are representative of one of at least three independent experiments (a-g) or one of three independent experiments (h,i).


Immunofluorescence microscopy of wild - type and RIP2 - deficient MEFs expressing Flag - tagged ΔN85 - ATG16L1 and HA - Nod1 ( a ) or HA - Nod2 ( b ) . Scale bars , 10 μm . Area outlined at far left is enlarged at right . Data are representative one of two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence microscopy of wild-type and RIP2-deficient MEFs expressing Flag-tagged ΔN85-ATG16L1 and HA-Nod1 (a) or HA-Nod2 (b). Scale bars, 10 μm. Area outlined at far left is enlarged at right. Data are representative one of two independent experiments.


( a ) Fluorescence microscopy of immortalized GFP - LC3 - transduced lymphoblasts from donors homozygous for the normal ATG16L1 * 300T allele ( 300T ) or risk allele ATG16L1 * 300A ( 300A ) , left unstimulated or treated for 2 h with rapamycin ( 50 μg / ml ) or for 4 h with MDP ( 20 μg / ml ) . Green , GFP - LC3 . Scale bar , 5 μm . ( b ) Induction of GFP - LC3 signals by rapamycin , MDP or Gram - positive peptidoglycan ( PG + ; 20 μg / ml ) in cells treated as described in a , presented relative to GFP - LC3 signals in unstimulated control cells . * P 0 . 05 ( t - test ) . Data represent three independent experiments ( error bars ( b ) , s . d . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Fluorescence microscopy of immortalized GFP-LC3-transduced lymphoblasts from donors homozygous for the normal ATG16L1*300T allele (300T) or risk allele ATG16L1*300A (300A), left unstimulated or treated for 2 h with rapamycin (50 μg/ml) or for 4 h with MDP (20 μg/ml). Green, GFP-LC3. Scale bar, 5 μm. (b) Induction of GFP-LC3 signals by rapamycin, MDP or Gram-positive peptidoglycan (PG+; 20 μg/ml) in cells treated as described in a, presented relative to GFP-LC3 signals in unstimulated control cells. *P 0.05 (t-test). Data represent three independent experiments (error bars (b), s.d.).


( a ) Immunofluorescence microscopy of HeLa cells transfected with HA - Nod2 ( red ) and Flag - ΔN85 - ATG16L1 ( green ) and infected for 10 - 20 min with S . flexneri M90T . Blue , DAPI . Arrows indicate colocalization of Nod2 and ATG16L1 around invading bacteria . Scale bars , 5 μm . Data are representative of one of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Immunofluorescence microscopy of HeLa cells transfected with HA-Nod2 (red) and Flag-ΔN85-ATG16L1(green) and infected for 10-20 min with S. flexneri M90T. Blue, DAPI. Arrows indicate colocalization of Nod2 and ATG16L1 around invading bacteria. Scale bars, 5 μm. Data are representative of one of three independent experiments.


( b ) Immunofluorescence microscopy of endogenous Nod2 and ATG16L1 in Mode - K cells and infected for 10 - 20 min with S . flexneri M90T . Blue , DAPI . Arrows indicate colocalization of Nod2 and ATG16L1 around invading bacteria . Scale bars , 5 μm . Data are representative of one of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) Immunofluorescence microscopy of endogenous Nod2 and ATG16L1 in Mode-K cells and infected for 10-20 min with S. flexneri M90T. Blue, DAPI. Arrows indicate colocalization of Nod2 and ATG16L1 around invading bacteria. Scale bars, 5 μm. Data are representative of one of three independent experiments.


( a , b ) Immunofluorescence microscopy of HeLa cells expressing Flag - ΔN85 - ATG16L1 ( green ; a ) or Myc - FL - ATG16L1 ( green ; b ) and HA - tagged Nod2 L1007fsinsC ( HA - Nod2 , 1007fs ) . Area outlined at left is enlarged in other images .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a,b) Immunofluorescence microscopy of HeLa cells expressing Flag-ΔN85-ATG16L1 (green; a) or Myc-FL-ATG16L1 (green; b) and HA-tagged Nod2 L1007fsinsC (HA-Nod2, 1007fs). Area outlined at left is enlarged in other images.


( c , d ) Immunofluorescence microscopy of HeLa cells expressing Flag - ΔN85 - ATG16L1 ( c ) or Myc - FL - ATG16L1 ( d ) together with HA - tagged Nod2fs ( red ) . Blue , DAPI . Scale bar , 8 μm .

(c,d) Immunofluorescence microscopy of HeLa cells expressing Flag-ΔN85-ATG16L1 (c) or Myc-FL-ATG16L1 (d) together with HA-tagged Nod2fs (red). Blue, DAPI. Scale bar, 8 μm.


( e ) Immunofluorescence microscopy of wild - type and Nod2fs - KI BMDMs transduced with GFP - LC3 ( green ) and infected for 0 . 5 h with S . flexneri M90T - RFP ( red ) , followed by incubation for 1 h with gentamicin . Arrows indicate no colocalization of Nod2 with ΔN85 - ATG16L1 or FL - ATG16L1 . Scale bar , 15 μm . ( f ) Quantification of the colocalization of GFP - LC3 signals with S . flexneri M90T - RFP in experiments as described in e . Data are representative of one of three independent experiments ( error bars ( f ) , s . d . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) Immunofluorescence microscopy of wild-type and Nod2fs-KI BMDMs transduced with GFP-LC3 (green) and infected for 0.5 h with S. flexneri M90T-RFP (red), followed by incubation for 1 h with gentamicin. Arrows indicate no colocalization of Nod2 with ΔN85-ATG16L1 or FL-ATG16L1. Scale bar, 15 μm. (f) Quantification of the colocalization of GFP-LC3 signals with S. flexneri M90T-RFP in experiments as described in e. Data are representative of one of three independent experiments (error bars (f), s.d.).


( A ) Assessment of vacuole ( arrows ) formation in neutrophils after stimulation with PMA ( II ) and rapamycin ( III ) , compared with control cells ( I : treated with PBS and serum ) ( magnification 1000 × ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(A) Assessment of vacuole (arrows) formation in neutrophils after stimulation with PMA (II) and rapamycin (III), compared with control cells (I: treated with PBS and serum) (magnification 1000×).


( B ) ( I ) Presence of late autophagosomes as observed with MDC staining in cells treated with PMA and rapamycin ( RAP ) compared with control cells ( PBS ) and cells treated with PMA and 3 ‐ MA ( magnification 400 and 1000 × ) . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . Data are representative of six independent experiments and are presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 : PMA ( bar 2 : 86 . 16 ± 6 . 49 % ) , rapamycin ( bar 3 : 65 . 83 ± 8 . 47 % ) treated cells , cells treated with PMA and 3 ‐ MA ( bar 4 : 9 . 17 ± 0 . 75 % ) and cells treated with rapamycin and 3 ‐ MA ( bar 5 : 8 . 5 ± 1 . 87 % ) compared with control conditions ( bar 1 : 15 . 67 ± 3 . 50 % ) . The percentage of cells with acidified autophagosomes in PMN treated with PMA and 3 ‐ MA ( bar 4 ) or rapamycin and 3 ‐ MA ( bar 5 ) was significantly lower ( p < 0 . 05 ) compared with cells treated with PMA ( bar 2 ) and rapamycin ( bar 3 ) , respectively .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O

(B) (I) Presence of late autophagosomes as observed with MDC staining in cells treated with PMA and rapamycin (RAP) compared with control cells (PBS) and cells treated with PMA and 3‐MA (magnification 400 and 1000×). (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. Data are representative of six independent experiments and are presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05: PMA (bar 2: 86.16±6.49%), rapamycin (bar 3: 65.83±8.47%) treated cells, cells treated with PMA and 3‐MA (bar 4: 9.17±0.75%) and cells treated with rapamycin and 3‐MA (bar 5: 8.5±1.87%) compared with control conditions (bar 1: 15.67±3.50%). The percentage of cells with acidified autophagosomes in PMN treated with PMA and 3‐MA (bar 4) or rapamycin and 3‐MA (bar 5) was significantly lower (p<0.05) compared with cells treated with PMA (bar 2) and rapamycin (bar 3), respectively.


( C ) Analysis of LC3B ‐ I to LC3B ‐ II conversion in cells treated with PMA ( lane IV ) and rapamycin ( lane II ) , as observed by LC3B immunoblotting . Effect of 3 ‐ MA in both PMA ( lane V ) and rapamycin ( lane I ) ‐ treated cells . Lane III : control cells , and lane VI : cells treated only with 3 ‐ MA . One representative out of six independent experiments is shown .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O

(C) Analysis of LC3B‐I to LC3B‐II conversion in cells treated with PMA (lane IV) and rapamycin (lane II), as observed by LC3B immunoblotting. Effect of 3‐MA in both PMA (lane V) and rapamycin (lane I)‐treated cells. Lane III: control cells, and lane VI: cells treated only with 3‐MA. One representative out of six independent experiments is shown.


( D ) Impact of PI ‐ 3K inhibition using LY294002 on LC3B conversion after PMA challenge ( lane III ) , ( lane I : control cells , lane II : PMA , lane IV : LY294002 only ) . One representative out of four independent experiments is shown .

O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(D) Impact of PI‐3K inhibition using LY294002 on LC3B conversion after PMA challenge (lane III), (lane I: control cells, lane II: PMA, lane IV: LY294002 only). One representative out of four independent experiments is shown.


( E ) Immunofluorescence confocal microscopy staining of LC3B . ( I ) Control cells treated with PBS and serum , ( II ) PMA ‐ treated cells . 1 . DAPI : blue , 2 . LC3B : red and 3 . merge . ( III ) Percentage of PMN with LC3B aggregates . Control conditions ( bar 1 : 17 . 7 ± 2 . 4 % ) , 3 ‐ MA ( bar 2 : 3 ± 1 . 01 % ) , PMA ( bar 3 : 69 ± 2 . 1 % ) , PMA and 3 ‐ MA ( bar 4 : 4 ± 1 . 9 % ) , rapamycin ( bar 5 : 56 ± 1 . 6 % ) . Data are representative of five independent experiments and presented as mean ± SD ; Student ' s t ‐ test , * p < 0 . 01 compared to the control ( bar 1 ) or the indicated comparision ( bar 3 vs 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Immunofluorescence confocal microscopy staining of LC3B. (I) Control cells treated with PBS and serum, (II) PMA‐treated cells. 1. DAPI: blue, 2. LC3B: red and 3. merge. (III) Percentage of PMN with LC3B aggregates. Control conditions (bar 1: 17.7±2.4%), 3‐MA (bar 2: 3±1.01%), PMA (bar 3: 69±2.1%), PMA and 3‐MA (bar 4: 4±1.9%), rapamycin (bar 5: 56±1.6%). Data are representative of five independent experiments and presented as mean±SD; Student's t‐test, *p<0.01 compared to the control (bar 1) or the indicated comparision (bar 3 vs 4).


ATG3 and LC3B gene expression pattern in neutrophils after stimulation with PMA or rapamycin . ATG3 mRNA expression analysis ( fold ) after PMA ( 2 . 44 ± 0 . 78 ) and rapamycin ( RAP : 1 . 81 ± 0 . 41 ) stimulation . LC3B gene alterations ( PMA : 4 . 98 ± 0 . 59 ) and rapamycin ( RAP : 1 . 7 ± 0 . 55 ) . Effect of 3 ‐ MA pre ‐ treatment , for both stimuli , was also assessed ( PMA + 3 ‐ MA : ATG3 : 1 . 36 ± 0 . 64 , LC3B : 1 . 5 ± 0 . 49 and rapamycin + 3 ‐ MA : ATG3 : 1 . 28 ± 0 . 46 , LC3B : 1 . 24 ± 0 . 57 ) . No other significant alteration in ATG genes mRNA levels was detected . Representative data of ATG7 mRNA expression analysis after treatment with PMA ( 1 . 21 ± 0 . 42 ) and rapamycin ( 0 . 94 ± 0 . 64 ) . Data are representative of six independent experiments and presented as mean relative expression ± SD . Wilcoxon matched ‐ pairs test , * p < 0 . 05 and n . s . = not significant compared with control conditions ( dot line ) as indicated in the figure . Relative expression ( folds ) of each gene is derived from substitution of DCT values ( normalized by GAPDH DCT value ) in 2DDCT equation 36 .

O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ATG3 and LC3B gene expression pattern in neutrophils after stimulation with PMA or rapamycin. ATG3 mRNA expression analysis (fold) after PMA (2.44±0.78) and rapamycin (RAP: 1.81±0.41) stimulation. LC3B gene alterations (PMA: 4.98±0.59) and rapamycin (RAP: 1.7±0.55). Effect of 3‐MA pre‐treatment, for both stimuli, was also assessed (PMA+3‐MA: ATG3: 1.36±0.64, LC3B: 1.5±0.49 and rapamycin+3‐MA: ATG3: 1.28±0.46, LC3B: 1.24±0.57). No other significant alteration in ATG genes mRNA levels was detected. Representative data of ATG7 mRNA expression analysis after treatment with PMA (1.21±0.42) and rapamycin (0.94±0.64). Data are representative of six independent experiments and presented as mean relative expression±SD. Wilcoxon matched‐pairs test, *p<0.05 and n.s.=not significant compared with control conditions (dot line) as indicated in the figure. Relative expression (folds) of each gene is derived from substitution of DCT values (normalized by GAPDH DCT value) in 2DDCT equation 36.


( A ) ( I ) MDC staining in neutrophils treated with LPS , LTA , peptidoglycan ( PGN ) , loxorubin ( LOX ) and R848 ( 400 × ) . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . LPS ( 50 ng / mL ) ( bar 2 : 69 . 67 ± 8 . 21 % ) , LTA ( bar 3 : 75 . 17 ± 9 . 80 % ) , peptidoglycan ( bar 4 : 77 ± 6 . 96 % ) , loxorubin ( bar 5 : 66 . 67 ± 5 . 61 % ) , R848 ( bar 6 : 69 . 5 ± 7 . 45 % ) , poly I : C ( bar 7 : 16 . 33 ± 3 . 14 % ) and flagellin ( bar 8 : 19 . 83 ± 5 . 16 % ) − treated cells compared with control conditions ( bar 1 : 14 . 5 ± 4 . 97 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched − pairs test , * p < 0 . 05 , n . s . = not significant .

O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) (I) MDC staining in neutrophils treated with LPS, LTA, peptidoglycan (PGN), loxorubin (LOX) and R848 (400×). (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. LPS (50 ng/mL) (bar 2: 69.67±8.21%), LTA (bar 3: 75.17±9.80%), peptidoglycan (bar 4: 77±6.96%), loxorubin (bar 5: 66.67±5.61%), R848 (bar 6: 69.5±7.45%), poly I:C (bar 7: 16.33±3.14%) and flagellin (bar 8: 19.83±5.16%)−treated cells compared with control conditions (bar 1: 14.5±4.97%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched−pairs test, *p<0.05, n.s.=not significant.


( B ) LC3B lipidation status of cells treated with LPS ( lane I ) , LTA ( lane II ) , peptidoglycan ( lane III ) , loxoribin ( lane IV ) and R848 ( lane V ) , detected by immunoblotting . One representative out of six independent experiments is shown .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

(B) LC3B lipidation status of cells treated with LPS (lane I), LTA (lane II), peptidoglycan (lane III), loxoribin (lane IV) and R848 (lane V), detected by immunoblotting. One representative out of six independent experiments is shown.


( C ) LC3B − II / LC3B - I ratio as obtained from integrated optical density measurements . LTA ( bar 2 : 1 . 75 ± 0 . 84 ) , peptidoglycan ( bar 3 : 1 . 82 ± 1 . 17 ) , LPS ( bar 4 : 1 . 9 ± 1 . 7 ) , loxorubin ( bar 5 : 1 . 32 ± 0 . 49 ) and R848 ( bar 6 : 1 . 4 ± 0 . 77 ) compared with control neutrophils ( bar 1 : 0 . 76 ± 0 . 30 ) . Data are representative of six independent experiments and are presented as mean ± SD . Wilcoxon matched − pairs test , * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) LC3B−II/LC3B-I ratio as obtained from integrated optical density measurements. LTA (bar 2: 1.75±0.84), peptidoglycan (bar 3: 1.82±1.17), LPS (bar 4: 1.9±1.7), loxorubin (bar 5: 1.32±0.49) and R848 (bar 6: 1.4±0.77) compared with control neutrophils (bar 1: 0.76±0.30). Data are representative of six independent experiments and are presented as mean±SD. Wilcoxon matched−pairs test, *p<0.05.


( D ) Analysis of LC3B gene expression levels in neutrophils treated with TLR agonists . LPS ( bar 1 : 2 . 27 ± 0 . 43 ) , peptidoglycan ( bar 2 : 2 . 07 ± 0 . 42 ) , LTA ( bar 3 : 2 . 1 ± 0 . 49 ) and R848 ( bar 4 : 1 . 89 ± 0 . 35 ) compared with medium − treated control cells . Dotted line represents LC3B mRNA levels of medium - treated control cells . Data are representative of six independent experiments and are presented as mean relative expression ± SD . Wilcoxon matched − pairs test , * p < 0 . 05 .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Analysis of LC3B gene expression levels in neutrophils treated with TLR agonists. LPS (bar 1: 2.27±0.43), peptidoglycan (bar 2: 2.07±0.42), LTA (bar 3: 2.1±0.49) and R848 (bar 4: 1.89±0.35) compared with medium−treated control cells. Dotted line represents LC3B mRNA levels of medium-treated control cells. Data are representative of six independent experiments and are presented as mean relative expression±SD. Wilcoxon matched−pairs test, *p<0.05.


( E ) Assessment of ATG gene expression levels in PMN after LPS stimulation ( 50 ng / mL ) . ATG3 ( bar 1 : 2 . 94 ± 0 . 52 ) , ATG4 ( bar 2 : 1 . 72 ± 0 . 42 ) , ATG5 ( bar 3 : 1 . 84 ± 0 . 34 ) , ATG6 ( bar 4 : 1 . 8 ± 0 . 3 ) , ATG7 ( bar 5 : 3 . 13 ± 0 . 64 ) and LC3B ( bar 6 : 2 . 27 ± 0 . 43 ) compared with control cells . Dotted line represents mRNA levels of medium - treated cells . Data are representative of six independent experiments and are presented as mean relative expression ± SD . Wilcoxon matched − pairs test , * p < 0 . 05 .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Assessment of ATG gene expression levels in PMN after LPS stimulation (50 ng/mL). ATG3 (bar 1: 2.94±0.52), ATG4 (bar 2: 1.72±0.42), ATG5 (bar 3: 1.84±0.34), ATG6 (bar 4: 1.8±0.3), ATG7 (bar 5: 3.13±0.64) and LC3B (bar 6: 2.27±0.43) compared with control cells. Dotted line represents mRNA levels of medium-treated cells. Data are representative of six independent experiments and are presented as mean relative expression±SD. Wilcoxon matched−pairs test, *p<0.05.


( A ) ( I ) Formation of acidified autophagosomes after stimulation with 10 ng / mL of IL ‐ 1β , compared with medium ‐ treated control cells . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . IL ‐ 1β ‐ treated cells ( bar 2 : 65 . 17 ± 10 . 23 % ) compared with control conditions ( bar 1 : 15 . 83 ± 6 . 08 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) (I) Formation of acidified autophagosomes after stimulation with 10 ng/mL of IL‐1β, compared with medium‐treated control cells. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. IL‐1β‐treated cells (bar 2: 65.17±10.23%) compared with control conditions (bar 1: 15.83±6.08%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05.


( B ) LC3B conversion in neutrophils treated with 10 ng / mL ( lane II ) and 100 ng / mL ( lane III ) of IL ‐ 1β . ( lane I : medium ‐ treated control cells ) . One representative out of five independent experiments is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) LC3B conversion in neutrophils treated with 10 ng/mL (lane II) and 100 ng/mL (lane III) of IL‐1β. (lane I: medium‐treated control cells). One representative out of five independent experiments is shown.


( A ) Effect of 3 ‐ MA on neutrophil phagocytic capacity of opsonized E . coli as observed by flow cytometry . Percentage of phagocytosing neutrophils after 3 ‐ MA pretreatment ( III ) compared with cells incubated with opsonized bacteria ( II ) . Data are representative of six independent experiments and are presented as mean ± SD , ( I : control unstimulated group ) , ( IV ) MFI representation ( bar 1 : control : 1 ± 1 . 9 , bar 2 : E . coli : 387 . 7 ± 33 . 3 , bar 3 : E . coli + 3 ‐ MA : 25 . 13 ± 18 , Wilcoxon matched ‐ pairs test , * p < 0 . 05 ; bar 2 compared with bar 1 and bar 3 compared to bar 2 ) .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Effect of 3‐MA on neutrophil phagocytic capacity of opsonized E. coli as observed by flow cytometry. Percentage of phagocytosing neutrophils after 3‐MA pretreatment (III) compared with cells incubated with opsonized bacteria (II). Data are representative of six independent experiments and are presented as mean±SD, (I: control unstimulated group), (IV) MFI representation (bar 1: control: 1±1.9, bar 2: E. coli: 387.7±33.3, bar 3: E. coli+3‐MA: 25.13±18, Wilcoxon matched‐pairs test, *p<0.05; bar 2 compared with bar 1 and bar 3 compared to bar 2).


( B ) ( I ) Presence of acidified structures in E . coli ‐ phagocytosing neutrophils and medium ‐ treated control cells . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . E . coli ‐ phagocytosing cells ( bar 2 : 79 . 33 ± 7 . 15 % ) compared with control conditions ( bar 1 : 14 . 17 ± 4 . 79 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) (I) Presence of acidified structures in E. coli‐phagocytosing neutrophils and medium‐treated control cells. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. E. coli‐phagocytosing cells (bar 2: 79.33±7.15%) compared with control conditions (bar 1: 14.17±4.79%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05. (


( C ) LC3B conversion analysis in E . coli ‐ phagocytosing neutrophils ( lane II ) , control neutrophils ( lane I ) and E . coli ‐ phagocytosing neutrophils after 3 ‐ MA pretreatment ( lane III ) . One representative out of six independent experiments is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(C) LC3B conversion analysis in E. coli‐phagocytosing neutrophils (lane II), control neutrophils (lane I) and E. coli‐phagocytosing neutrophils after 3‐MA pretreatment (lane III). One representative out of six independent experiments is shown.


( D ) Effect of 3 ‐ MA administration to E . coli ‐ phagocytosing neutrophils 15 min after the initiation of phagocytosis ( lane III ) as detected by LC3B conversion analysis . Lane II represents E . coli ‐ phagocytosing neutrophils and lane I control neutrophils . One representative out of four independent experiments is shown .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Effect of 3‐MA administration to E. coli‐phagocytosing neutrophils 15 min after the initiation of phagocytosis (lane III) as detected by LC3B conversion analysis. Lane II represents E. coli‐phagocytosing neutrophils and lane I control neutrophils. One representative out of four independent experiments is shown.


( E ) ( I ) Percentage of neutrophils with LC3B punctuated structures . E . coli ( bar 1 : 84 ± 2 . 2 % ) compared with E . coli + 3 ‐ MA ( bar 2 : 4 ± 1 . 95 % ) . Data are representative of six independent experiments and presented as mean ± SD . Wilcoxon matched ‐ pairs test , * p < 0 . 05 . ( II ) Formation of LC3B puncta in phagocytosing neutrophils and ( III ) in cells treated with 3 ‐ MA before incubation with opsonized E . coli , as observed by immunofluorescence confocal microscopy ( 1 . LC3B : red ; 2 . FITC conjugated E . coli : green ; 3 . DAPI : blue ; and 4 . merge ) . Solid line arrows point the colocalization of LC3B puncta with E . coli bacteria . Dashed line arrows point LC3B aggregates that are not colocalized with E . coli .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) (I) Percentage of neutrophils with LC3B punctuated structures. E. coli (bar 1: 84±2.2%) compared with E. coli+3‐MA (bar 2: 4±1.95%). Data are representative of six independent experiments and presented as mean±SD. Wilcoxon matched‐pairs test, *p<0.05. (II) Formation of LC3B puncta in phagocytosing neutrophils and (III) in cells treated with 3‐MA before incubation with opsonized E. coli, as observed by immunofluorescence confocal microscopy (1. LC3B: red; 2. FITC conjugated E. coli: green; 3. DAPI: blue; and 4. merge). Solid line arrows point the colocalization of LC3B puncta with E. coli bacteria. Dashed line arrows point LC3B aggregates that are not colocalized with E. coli.


Alterations in ATG gene expression after phagocytosis of opsonized E . coli . ATG3 ( 2 . 05 ± 0 . 27 ) , ATG4 ( 2 . 20 ± 0 . 27 ) , ATG5 ( 2 . 89 ± 0 . 42 ) , ATG6 ( 2 . 04 ± 0 . 40 ) , ATG7 ( 1 . 98 ± 0 . 30 ) and LC3B ( 2 . 46 ± 0 . 39 ) gene expression ( fold ) in E . coli ‐ phagocytosing neutrophils compared with control cells . Dotted line represents mRNA expression levels of control cells . Data are representative of six independent experiments and are presented as mean relative expression ± SD Wilcoxon matched ‐ pairs test , * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Alterations in ATG gene expression after phagocytosis of opsonized E. coli. ATG3 (2.05±0.27), ATG4 (2.20±0.27), ATG5 (2.89±0.42), ATG6 (2.04±0.40), ATG7 (1.98±0.30) and LC3B (2.46±0.39) gene expression (fold) in E. coli‐phagocytosing neutrophils compared with control cells. Dotted line represents mRNA expression levels of control cells. Data are representative of six independent experiments and are presented as mean relative expression±SD Wilcoxon matched‐pairs test, *p<0.05.


( A ) LC3B conversion status after pretreatment with the ROS scavenger BHA prior to PMA or E . coli stimulation ( lane I : PMA , lane II : PMA + BHA , lane III : E . coli , lane IV : E . coli + BHA , lane V : control cells ) . One representative out of four independent experiments is shown .

O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

(A) LC3B conversion status after pretreatment with the ROS scavenger BHA prior to PMA or E. coli stimulation (lane I: PMA, lane II: PMA+BHA, lane III: E. coli, lane IV: E. coli+BHA, lane V: control cells). One representative out of four independent experiments is shown.


( B ) LC3B conversion status after pretreatment with the NADPH oxidase inhibitor DPI in E . coli − phagocytosing neutrophils ( lane I : PBS , lane II : E . coli , lane III : E . coli + DPI ) . One representative out of four independent experiments is shown .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

(B) LC3B conversion status after pretreatment with the NADPH oxidase inhibitor DPI in E. coli−phagocytosing neutrophils (lane I: PBS, lane II: E. coli, lane III: E. coli+DPI). One representative out of four independent experiments is shown.


( C ) ( I ) Effect of NADPH ‐ oxidase inhibition with DPI in the formation of acidified autophagosomes in E . coli ‐ phagocytosing neutrophils . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . E . coli − phagocytosing PMN ( 77 . 17 ± 8 . 33 % ) and PMN treated with DPI before incubation with opsonized E . coli ( 21 . 67 ± 4 . 37 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) (I) Effect of NADPH‐oxidase inhibition with DPI in the formation of acidified autophagosomes in E. coli‐phagocytosing neutrophils. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. E. coli−phagocytosing PMN (77.17±8.33%) and PMN treated with DPI before incubation with opsonized E. coli (21.67±4.37%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05


( D ) ( I ) Assessment of formation of acidified autophagosomes in neutrophils pretreated with BNP before stimulation with peptidoglycan ( PGN ) , compared with cells treated with PGN . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . PGN ‐ treated neutrophils ( 74 . 83 ± 9 . 09 % ) and neutrophils pretreated with BNP before the addition of PGN ( 17 . 33 ± 2 . 07 % ) compared with control conditions ( 14 . 67 ± 3 . 20 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 , n . s . = not significant :

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) (I) Assessment of formation of acidified autophagosomes in neutrophils pretreated with BNP before stimulation with peptidoglycan (PGN), compared with cells treated with PGN. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. PGN‐treated neutrophils (74.83±9.09%) and neutrophils pretreated with BNP before the addition of PGN (17.33±2.07%) compared with control conditions (14.67±3.20%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05, n.s.=not significant:


( E ) Alterations in LC3B conversion in BNP pretreated , peptidoglycan ‐ stimulated neutrophils ( lane III ) , ( lane I : medium ‐ treated cells , lane II : peptidoglycan ‐ treated cells ) . One representative out of four independent experiments is shown .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Alterations in LC3B conversion in BNP pretreated, peptidoglycan‐stimulated neutrophils (lane III), (lane I: medium‐treated cells, lane II: peptidoglycan‐treated cells). One representative out of four independent experiments is shown.


( F ) Effect of PI3K inhibition with 3 ‐ MA in the percentage of PMA ‐ induced oxidizing cells . PMA ( 99 . 41 ± 0 . 33 % ) and PMA + 3 ‐ MA ( 15 . 82 ± 3 . 97 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Effect of PI3K inhibition with 3‐MA in the percentage of PMA‐induced oxidizing cells. PMA (99.41±0.33%) and PMA+3‐MA (15.82±3.97%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05.


( A ) Recombinant proteins were mixed , as indicated , in the presence ( lane 5 ) or absence ( lane 4 ) of ATP and incubated for 1 h at 30 ° C before being separated by SDS - PAGE and analyzed by western blotting using the α - His antibody . Components present ( + ) or absent ( − ) from the assay are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Recombinant proteins were mixed, as indicated, in the presence (lane 5) or absence (lane 4) of ATP and incubated for 1 h at 30°C before being separated by SDS-PAGE and analyzed by western blotting using the α-His antibody. Components present (+) or absent (−) from the assay are indicated.


( B ) Recombinant proteins were mixed , as indicated , and incubated as above . The reactions were stopped , subjected to SDS - PAGE and stained with Coomassie Blue .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Recombinant proteins were mixed, as indicated, and incubated as above. The reactions were stopped, subjected to SDS-PAGE and stained with Coomassie Blue.


( C ) Recombinant proteins were mixed , as indicated , incubated as above and analyzed with α - His antibody .

O O O O O O O O O O O O O O O O O O O O O O

(C) Recombinant proteins were mixed, as indicated, incubated as above and analyzed with α-His antibody.


( A ) The occurrence of mC - ATG5 puncta in WT L . major promastigotes expressing mC - ATG5 incubated in nutrient - rich medium at log phase at 26 ° C .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) The occurrence of mC-ATG5 puncta in WT L. major promastigotes expressing mC-ATG5 incubated in nutrient-rich medium at log phase at 26°C.


( B ) The multiplicity of puncta in promastigotes expressing mC - ATG5 after starvation for up to 1 h in PBS at 26 ° C . The time ranges indicated reflect the starvation period together with the 30 min period during which the observations on the microscope were undertaken .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) The multiplicity of puncta in promastigotes expressing mC-ATG5 after starvation for up to 1 h in PBS at 26°C. The time ranges indicated reflect the starvation period together with the 30 min period during which the observations on the microscope were undertaken.