( H ) FTMT mRNA expression was quantified for Huh7 cells by real - time RT - PCR before and after DFP ( 1 . 0 mM ) treatment with or without SP1 knockdown by siRNA ( n = 5 , biological replicates ) . mRNA expression of FTMT level was normalized to GAPDH . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . NC : negative control siRNA , * * : P < 0 . 01 .

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(H) FTMT mRNA expression was quantified for Huh7 cells by real-time RT-PCR before and after DFP (1.0 mM) treatment with or without SP1 knockdown by siRNA (n=5, biological replicates). mRNA expression of FTMT level was normalized to GAPDH. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. NC: negative control siRNA, **: P<0.01.


( I ) Immunoblots for SP1 using the cytoplasmic fraction and nuclear fraction before and after DFP ( 1 . 0 mM ) treatment . β - actin and Lam B were used as loading control for the cytoplasmic and nuclear fractions , respectively .

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(I) Immunoblots for SP1 using the cytoplasmic fraction and nuclear fraction before and after DFP (1.0 mM) treatment. β-actin and Lam B were used as loading control for the cytoplasmic and nuclear fractions, respectively.


( J ) Immunoblots for HIF1α using Huh7 cells and HepG2 cells before and after DFP ( 0 . 1 mM ) treatment . β - actin was used as the loading control .

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(J) Immunoblots for HIF1α using Huh7 cells and HepG2 cells before and after DFP (0.1 mM) treatment. β-actin was used as the loading control.


( K ) Immunoblots for FTMT , HIF1α and SP1 using the mitochondrial fraction lysate and whole cell lysate of Huh7 cells before and after DFP ( 1 . 0 mM ) treatment with or without HIF1α or SP1 knockdown by siRNA . HSP60 and β - actin were used as loading controls for the mitochondrial fraction and whole cell lysates , respectively .

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(K) Immunoblots for FTMT, HIF1α and SP1 using the mitochondrial fraction lysate and whole cell lysate of Huh7 cells before and after DFP (1.0 mM) treatment with or without HIF1α or SP1 knockdown by siRNA. HSP60 and β-actin were used as loading controls for the mitochondrial fraction and whole cell lysates, respectively.


( A ) Immunoblots for mitochondrial ferritin ( FTMT ) with HSP60 as the loading control using the mitochondrial fraction of Huh7 cells and HepG2 cells ( upper panels ) and the quantification of mitophagy using mt - mKeima transfection and FACS analysis ( n = 4 , biological replicates ) ( lower panels ) before and after DFP ( 1 . 0 mM ) treatment with or without FTMT knockdown by siRNA . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . NC : negative control siRNA , * * : P < 0 . 01 .

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

(A) Immunoblots for mitochondrial ferritin (FTMT) with HSP60 as the loading control using the mitochondrial fraction of Huh7 cells and HepG2 cells (upper panels) and the quantification of mitophagy using mt-mKeima transfection and FACS analysis (n=4, biological replicates) (lower panels) before and after DFP (1.0 mM) treatment with or without FTMT knockdown by siRNA. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. NC: negative control siRNA, **: P<0.01.


( B ) Immunofluorescence staining for LC3 ( red ) , the mitochondrial marker Tom20 ( green ) and FTMT ( blue ) in Huh7 cells before and after DFP ( 1 . 0 mM ) treatment with or without FTMT knockdown by siRNA . FTMT colocalize with LC3 at the mitochondria ( white puncta indicated by arrows ) . Boxed areas are enlarged below . Scale bar : 5

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(B) Immunofluorescence staining for LC3 (red), the mitochondrial marker Tom20 (green) and FTMT (blue) in Huh7 cells before and after DFP (1.0 mM) treatment with or without FTMT knockdown by siRNA. FTMT colocalize with LC3 at the mitochondria (white puncta indicated by arrows). Boxed areas are enlarged below. Scale bar: 5 μm


( C ) Immunoblots for mitochondrial ferritin ( FTMT ) following FTMT knockdown in the absence or presence of DFP using the mitochondrial fraction of Huh7 cells and those transfected with FTMT siRNA - resistant FTMT ( left panel ) . HSP60 as the loading control . Those cells were subjected to the quantification of mitophagy using mt - mKeima transfection and FACS analysis before and after DFP ( 1 . 0 mM ) treatment ( n = 5 , biological replicates ) ( right panel ) . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * * * : P < 0 . 001 .

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(C) Immunoblots for mitochondrial ferritin (FTMT) following FTMT knockdown in the absence or presence of DFP using the mitochondrial fraction of Huh7 cells and those transfected with FTMT siRNA-resistant FTMT (left panel). HSP60 as the loading control. Those cells were subjected to the quantification of mitophagy using mt-mKeima transfection and FACS analysis before and after DFP (1.0 mM) treatment (n=5, biological replicates) (right panel). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. ***: P<0.001.


( D ) Super resolution images of immunofluorescence staining for LC3 ( red ) , FTMT ( green ) and Tom20 ( blue ) in FTMT knockdown - Huh7 cells transfected with FTMT - GFP or FTMT siRNA - resistant FTMT - GFP before and after DFP ( 1 . 0 mM ) treatment . White arrows indicate colocalization of LC3 , FTMT , and Tom20 .

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(D) Super resolution images of immunofluorescence staining for LC3 (red), FTMT (green) and Tom20 (blue) in FTMT knockdown-Huh7 cells transfected with FTMT-GFP or FTMT siRNA-resistant FTMT-GFP before and after DFP (1.0 mM) treatment. White arrows indicate colocalization of LC3, FTMT, and Tom20.


( A ) Extracted proteins from Huh7 cells that were untreated or treated with DFP were mixed with anti - FTMT - coupled magnetic beads and immunoblotted with the indicated antibodies ( anti - NCOA4 or anti - LC3 ) .

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(A) Extracted proteins from Huh7 cells that were untreated or treated with DFP were mixed with anti-FTMT-coupled magnetic beads and immunoblotted with the indicated antibodies (anti-NCOA4 or anti-LC3).


( B ) GST pulldown assay using recombinant GST - NCOA4 mixed with recombinant His - FTMT . Bound FTMT was analyzed by immunoblotting using either anti - FTMT or anti - His antibodies .

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(B) GST pulldown assay using recombinant GST-NCOA4 mixed with recombinant His-FTMT. Bound FTMT was analyzed by immunoblotting using either anti-FTMT or anti-His antibodies.


( C ) Immunofluorescence staining for Tom20 ( red ) , NCOA4 ( green ) and FTMT ( blue ) in Huh7 cells before and after DFP ( 1 mM ) treatment . Boxed areas are enlarged on the right . The white puncta indicated by white arrows show the colocalization of FTMT , NCOA4 and Tom20 . Scale bar : 10 μm and 5 μm in enlarged images .

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(C) Immunofluorescence staining for Tom20 (red), NCOA4 (green) and FTMT (blue) in Huh7 cells before and after DFP (1 mM) treatment. Boxed areas are enlarged on the right. The white puncta indicated by white arrows show the colocalization of FTMT, NCOA4 and Tom20. Scale bar: 10 μm and 5 μm in enlarged images.


( D ) Super - resolution images of immunofluorescence staining for Tom20 ( blue ) , NCOA4 ( red ) and FTMT ( green ) in Huh7 cells after DFP ( 1 mM ) treatment , using the single - molecule fluorescence microscope . The white arrows indicate colocalization of these proteins .

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(D) Super-resolution images of immunofluorescence staining for Tom20 (blue), NCOA4 (red) and FTMT (green) in Huh7 cells after DFP (1 mM) treatment, using the single-molecule fluorescence microscope. The white arrows indicate colocalization of these proteins.


( E ) Immunofluorescence staining for Tom20 ( blue ) , FTMT - GFP ( green ) , recombinant FTMTR82A - GFP ( green ) , NCOA4 - mCherry ( red ) and NCOA4I489A / W497A - mCherry ( red ) in Huh7 cells . Boxed areas are enlarged on the right . The white puncta pointed by white arrows in the overlaid picture indicate the colocalization of Tom20 , FTMT - GFP and NCOA4 - mCherry . The yellow puncta shown by white arrows in the overlaid picture indicate the colocalization of FTMT - GFP and NCOA4 - mCherry . Scale bar : 10 μm and 5 μm in enlarged images .

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(E) Immunofluorescence staining for Tom20 (blue), FTMT-GFP (green), recombinant FTMTR82A-GFP (green), NCOA4-mCherry (red) and NCOA4I489A/W497A-mCherry (red) in Huh7 cells. Boxed areas are enlarged on the right. The white puncta pointed by white arrows in the overlaid picture indicate the colocalization of Tom20, FTMT-GFP and NCOA4-mCherry. The yellow puncta shown by white arrows in the overlaid picture indicate the colocalization of FTMT-GFP and NCOA4-mCherry. Scale bar: 10 μm and 5 μm in enlarged images.


( F ) Extracted proteins from Huh7 cells expressing the recombinant proteins as indicated were mixed with anti - GFP - coupled magnetic beads and immunoblotted with anti - mCherry and anti - GFP antibodies . Co - immunoprecipitation revealed a specific interaction of NCOA4 - mCherry with FTMT - GFP or FTMTD57A - GFP , which has a substitution of alanine ( A ) for aspartic acid ( D ) at position 57 in the FTMT mitochondrial leader sequence ( lanes 1 and 3 ) .

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(F) Extracted proteins from Huh7 cells expressing the recombinant proteins as indicated were mixed with anti-GFP-coupled magnetic beads and immunoblotted with anti-mCherry and anti-GFP antibodies. Co-immunoprecipitation revealed a specific interaction of NCOA4-mCherry with FTMT-GFP or FTMTD57A-GFP, which has a substitution of alanine (A) for aspartic acid (D) at position 57 in the FTMT mitochondrial leader sequence (lanes 1 and 3).


( G ) Following DFP ( 1 . 0 mM ) or ammonium iron sulfate ( FAS ) treatment as indicated , extracted proteins from Huh7 cells expressing FTMT - GFP and NCOA4 - mCherry were mixed with anti - GFP - coupled magnetic beads and immunoblotted with anti - mCherry and anti - GFP antibodies .

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(G) Following DFP (1.0 mM) or ammonium iron sulfate (FAS) treatment as indicated, extracted proteins from Huh7 cells expressing FTMT-GFP and NCOA4-mCherry were mixed with anti-GFP-coupled magnetic beads and immunoblotted with anti-mCherry and anti-GFP antibodies.


( A ) Flowcytometric analysis of the mitochondrial membrane potential of the isolated mitochondria . The mitochondria isolated from Huh7 cells with CCCP treatment and with or without DFP treatment were incubated with JC - 1 for 1 h at 30 ° C and subjected to flow cytometric analysis ( n = 4 , biological replicates ) by excitation with a blue laser ( 488 nm ) with simultaneous emission assessment using a 585 / 42 detector for JC - 1 red and a 530 / 30 for JC - 1 green . The trapezoid areas show the population of depolarized mitochondria . Quantification of the proportion of depolarized mitochondria in Huh7 cells with or without DFP treatment is shown in the lower panel . The central horizontal bar and the error bards indicate mean ± SD . Two sample t test was used for statistical analysis . * * : P < 0 . 01 .

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(A) Flowcytometric analysis of the mitochondrial membrane potential of the isolated mitochondria. The mitochondria isolated from Huh7 cells with CCCP treatment and with or without DFP treatment were incubated with JC-1 for 1 h at 30°C and subjected to flow cytometric analysis (n=4, biological replicates) by excitation with a blue laser (488 nm) with simultaneous emission assessment using a 585/42 detector for JC-1 red and a 530/30 for JC-1 green. The trapezoid areas show the population of depolarized mitochondria. Quantification of the proportion of depolarized mitochondria in Huh7 cells with or without DFP treatment is shown in the lower panel. The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. **: P<0.01.


( B ) The oxygen consumption rate was measured using a Seahorse XF24 Extracellular Flux Analyzer ( n = 4 , technical replicates ) . Huh7 cells were incubated with 1 mM of DFP for different lengths of time ( 0 , 3 , 6 , 18 , and 24 h ) . Oxygen consumption was measured , and the respiration rate was analyzed by injecting of 1μM oligomycin , 1 μM carbonyl cyanide 4 - ( trifluoromethoxy ) phenylhydrazone ( FCCP ) and 10 μM rotenone / antimycin A . OCR : oxygen consumption rate . The central point and the error bards indicate mean ± SD .

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(B) The oxygen consumption rate was measured using a Seahorse XF24 Extracellular Flux Analyzer (n=4, technical replicates). Huh7 cells were incubated with 1 mM of DFP for different lengths of time (0, 3, 6, 18, and 24 h). Oxygen consumption was measured, and the respiration rate was analyzed by injecting of 1μM oligomycin, 1 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and 10 μM rotenone/antimycin A. OCR: oxygen consumption rate. The central point and the error bards indicate mean ± SD.


( C ) Immunofluorescence staining for FTMT - GFP ( green ) and LC3 ( red ) in Huh7 cells transfected with FTMT - GFP . The proportion of colocalization was quantified for four randomly selected areas ( technical replicates ) using ImageJ software version 1 . 46 ( NIH , Bethesda , MD ) . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * * : P < 0 . 01 .

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(C) Immunofluorescence staining for FTMT-GFP (green) and LC3 (red) in Huh7 cells transfected with FTMT-GFP. The proportion of colocalization was quantified for four randomly selected areas (technical replicates) using ImageJ software version 1.46 (NIH, Bethesda, MD). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. **: P<0.01.


( D ) Immunofluorescence staining for Ferritin - GFP heavy chain ( FTH1 ) ( green ) and LC3 ( red ) in Huh7 cells transfected with FTH1 - GFP . The proportion of colocalization was quantified as described in ( C ) . * : P < 0 . 05 , * * : P < 0 . 01 .

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(D) Immunofluorescence staining for Ferritin-GFP heavy chain (FTH1) (green) and LC3 (red) in Huh7 cells transfected with FTH1-GFP. The proportion of colocalization was quantified as described in (C). *: P<0.05, **: P<0.01.


( E ) Immunoblots for the precursor form and processed form of FTMT - GFP , endogenous FTMT , NCOA4 , LC3 , and Tom20 using the mitochondrial fractions of Huh7 cells expressing FTMT - GFP with or without digestion with proteinase K in the presence or absence of DFP . mtHSP70 was used as the loading control .

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(E) Immunoblots for the precursor form and processed form of FTMT-GFP, endogenous FTMT, NCOA4, LC3, and Tom20 using the mitochondrial fractions of Huh7 cells expressing FTMT-GFP with or without digestion with proteinase K in the presence or absence of DFP. mtHSP70 was used as the loading control.


( F ) Immunoblots for FTMT , VDAC , and mtHSP70 using the mitochondrial membrane from Huh7 cells with or without DFP treatment . The mitochondrial membrane was fractionated from Huh7 cells by means of sodium carbonate treatment . mtHSP70 was used as the non - membrane - integral protein control and VDAC as the membrane - integral protein control . mtHSP70 at the right - hand side in the third lane indicates the positive control obtained from the mitochondrial fraction of Huh7 cells without DFP treatment .

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(F) Immunoblots for FTMT, VDAC, and mtHSP70 using the mitochondrial membrane from Huh7 cells with or without DFP treatment. The mitochondrial membrane was fractionated from Huh7 cells by means of sodium carbonate treatment. mtHSP70 was used as the non-membrane-integral protein control and VDAC as the membrane-integral protein control. mtHSP70 at the right-hand side in the third lane indicates the positive control obtained from the mitochondrial fraction of Huh7 cells without DFP treatment.


( A ) Immunofluorescence staining for the polarized mitochondria ( red ) , Tom20 ( blue ) , and LC3 ( green ) in Huh7 cells untreated or treated with DFP ( 1 mM ) for 24 h , or with CCCP ( 30 μM ) for 6 h . Boxed areas are enlarged on the right . Scale bar : 10 μm , and 5 μm in enlarged images .

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(A) Immunofluorescence staining for the polarized mitochondria (red), Tom20 (blue), and LC3 (green) in Huh7 cells untreated or treated with DFP (1 mM) for 24 h, or with CCCP (30 μM) for 6 h. Boxed areas are enlarged on the right. Scale bar: 10 μm, and 5 μm in enlarged images.


( B ) Immunofluorescence staining for the polarized mitochondria ( red ) , Tom20 ( blue ) , LC3 ( blue ) , and FTMTD57A ( green ) using Huh7 cells expressing FTMTD57A . Boxed areas are enlarged on the right . The white puncta in overlaid pictures indicate the colocalization of the polarized mitochondria , Tom20 and FTMTD57A or the colocalization of the polarized mitochondria , FTMTD57A , and LC3 . Scale bar : 10 μm , and 5 μm in enlarged images .

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(B) Immunofluorescence staining for the polarized mitochondria (red), Tom20 (blue), LC3 (blue), and FTMTD57A (green) using Huh7 cells expressing FTMTD57A. Boxed areas are enlarged on the right. The white puncta in overlaid pictures indicate the colocalization of the polarized mitochondria, Tom20 and FTMTD57A or the colocalization of the polarized mitochondria, FTMTD57A, and LC3. Scale bar: 10 μm, and 5 μm in enlarged images.


( C ) STAM mice were fed a high - fat diet and orally administered 0 . 0375 mg / g body weight of DFP , 0 . 075 mg / g body weight of DFP or distilled water from 4 weeks of age to 16 weeks of age ( n = 6 ) . Arrows indicate liver tumors . Scale bar : 1 . 0 cm . The number and maximum size of liver tumors were compared among the three groups . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * : P < 0 . 05 , * * : P < 0 . 01 .

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(C) STAM mice were fed a high-fat diet and orally administered 0.0375 mg/g body weight of DFP, 0.075 mg/g body weight of DFP or distilled water from 4 weeks of age to 16 weeks of age (n=6). Arrows indicate liver tumors. Scale bar: 1.0 cm. The number and maximum size of liver tumors were compared among the three groups. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01.


( D ) Histology of the nontumorous liver ( H & E and Sirius red stain , original magnification x40 ) and superoxide anion production in the livers of STAM mice fed a high - fat diet with or without DFP ( 0 . 075 mg / g ) administration . Frozen liver sections of mice were stained with dihydroethidium for assessment of superoxide anion . Scale bar : 100 μm .

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(D) Histology of the nontumorous liver (H&E and Sirius red stain, original magnification x40) and superoxide anion production in the livers of STAM mice fed a high-fat diet with or without DFP (0.075 mg/g) administration. Frozen liver sections of mice were stained with dihydroethidium for assessment of superoxide anion. Scale bar: 100 μm.


( E ) Histology of the hepatocellular carcinoma using H & E staining in STAM mice fed a high - fat diet . The boxed area is enlarged on the right ( x40 in left panel and x400 in right panel ) . The tumor has increased cell density , an increased ratio of nucleus to cytoplasm , and thickened trabeculae Scale bar : 100 μm .

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(E) Histology of the hepatocellular carcinoma using H&E staining in STAM mice fed a high-fat diet. The boxed area is enlarged on the right (x40 in left panel and x400 in right panel). The tumor has increased cell density, an increased ratio of nucleus to cytoplasm, and thickened trabeculae Scale bar: 100 μm.


( F ) Electron microscopic images of livers in control ( C57BL / 6 ) and DMBA + HFD mice that were orally administered 0 . 075 mg / g of DFP or distilled water . Scale bars : 2 . 0 μm . The boxed areas ( x 1 , 500 ) are enlarged below ( x6 , 000 on left and x10 , 000 on right ) . The white arrow indicates mitophagosome - like structures . The mean number of mitophagosome - like structures / 100 μm2 was compared for four randomly selected areas ( technical replicates ) among the four groups ( control , DMBA + HFD mice treated with 0 , 0 . 0375 or 0 . 075 mg / g of DFP ) ( n = 6 ) . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * : P < 0 . 05 .

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(F) Electron microscopic images of livers in control (C57BL/6) and DMBA + HFD mice that were orally administered 0.075 mg/g of DFP or distilled water. Scale bars: 2.0 μm. The boxed areas (x 1,500) are enlarged below (x6,000 on left and x10,000 on right). The white arrow indicates mitophagosome-like structures. The mean number of mitophagosome-like structures/100 μm2 was compared for four randomly selected areas (technical replicates) among the four groups (control, DMBA + HFD mice treated with 0, 0.0375 or 0.075 mg/g of DFP) (n=6). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05.


( A ) Immunoblots for FTMT , frataxin , mitoferrin , HSP60 , FTH1 and TfR using the hepatic mitochondrial fraction lysate and whole liver lysate in control ( C57BL / 6J ) mice and STAM mice treated with DFP ( 0 , 0 . 0375 , or 0 . 075 mg / g body weight ) ( n = 6 ) . The expression of FTMT , frataxin , mitoferrin , FTH1 , and TfR were normalized to HSP60 or β - actin , respectively . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * : P < 0 . 05 , * * : P < 0 . 01 . ( B ) The mean number of mitophagosome - like structures / 100 μm2 from four randomly selected areas in the livers of DFP ( 0 . 075 mg / g body weight ) - administered mice were compared between control and FTMT siRNA - treated groups for STAM mice ( n = 6 ) and DMBA + HFD mice ( n = 6 ) . The central horizontal bar and the error bards indicate mean ± SD . Two sample t test was used for statistical analysis . NC : negative control siRNA , * : 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 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 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 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

(A) Immunoblots for FTMT, frataxin, mitoferrin, HSP60, FTH1 and TfR using the hepatic mitochondrial fraction lysate and whole liver lysate in control (C57BL/6J) mice and STAM mice treated with DFP (0, 0.0375, or 0.075 mg/g body weight) (n=6). The expression of FTMT, frataxin, mitoferrin, FTH1, and TfR were normalized to HSP60 or β-actin, respectively. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01. (B) The mean number of mitophagosome-like structures/100 μm2 from four randomly selected areas in the livers of DFP (0.075 mg/g body weight)-administered mice were compared between control and FTMT siRNA-treated groups for STAM mice (n=6) and DMBA + HFD mice (n=6). The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. NC: negative control siRNA, *: P<0.05.


( C ) Frozen liver sections of DFP ( 0 . 075 mg / g body weight ) - administered mice ( STAM mice and DMBA + HFD ) with or without FTMT knockdown were stained with dihydroethidium . Red fluorescence indicates the ROS production . Scale bar : 100 μm . siNC : negative control siRNA .

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

(C) Frozen liver sections of DFP (0.075 mg/g body weight)-administered mice (STAM mice and DMBA + HFD) with or without FTMT knockdown were stained with dihydroethidium. Red fluorescence indicates the ROS production. Scale bar: 100 μm. siNC: negative control siRNA.


( D , E ) STAM mice ( D ) were fed a high - fat diet without DFP treatment or orally administered with 0 . 075 mg / g body weight of DFP from the age of 4 weeks , injected with FTMT siRNA ( 10 μl / g body weight ) or negative control siRNA through the tail vein every 2 weeks for 8 weeks prior to sacrifice , and followed until 16 weeks of age ( n = 6 ) . DMBA + HFD mice ( E ) were fed a high - fat diet , treated with DFP and siRNA in the same manner with STAM mice , and followed until 30 weeks of age ( n = 6 ) . The number and maximum size of liver tumors were compared among the three groups ( control without DFP , negative control siRNA , and FTMT siRNA with DFP ) . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * : P < 0 . 05 , * * : P < 0 . 01 . Representative liver images are shown in the upper panel . Scale bar : 1 cm .

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 O O O O O O O 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E) STAM mice (D) were fed a high-fat diet without DFP treatment or orally administered with 0.075 mg/g body weight of DFP from the age of 4 weeks, injected with FTMT siRNA (10 μl/g body weight) or negative control siRNA through the tail vein every 2 weeks for 8 weeks prior to sacrifice, and followed until 16 weeks of age (n=6). DMBA + HFD mice (E) were fed a high-fat diet, treated with DFP and siRNA in the same manner with STAM mice, and followed until 30 weeks of age (n=6). The number and maximum size of liver tumors were compared among the three groups (control without DFP, negative control siRNA, and FTMT siRNA with DFP). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01. Representative liver images are shown in the upper panel. Scale bar: 1 cm.


( C - F ) C . Total fresh weight , D . Total dry weight , E . Primary root length and F . Lateral root density of 21 - day - old , non - colonized ( Mock ) and SA190 - colonized plants upon growth for 16 days on ½ MS or ½ MS + 25 % PEG . Boxplot shows data from three independent biological replicates ( technical replicates n = 18 - 24 ) where whiskers represents minimum and maximum values , " + " indicates mean and horizontal line represents median . Asterisks indicate the statistical differences based on the 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 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

(C-F) C. Total fresh weight, D. Total dry weight, E. Primary root length and F. Lateral root density of 21-day-old, non-colonized (Mock) and SA190-colonized plants upon growth for 16 days on ½ MS or ½ MS + 25% PEG. Boxplot shows data from three independent biological replicates (technical replicates n = 18-24) where whiskers represents minimum and maximum values, "+" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (*** p < 0.001).


( G , H ) G . Growth and H . Survival percentage of mock - and SA190 - colonized plants in jiffy pots grown upon watering for 2 weeks , no watering for 3 weeks and after one week of re - watering . All plots represent the mean of 3 biological replicates ( technical replicates n = 36 ) . Whiskers represents minimum and maximum values , " + " indicates mean and horizontal line represents median . Asterisks indicate the statistical differences based on the 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

(G, H) G. Growth and H. Survival percentage of mock- and SA190-colonized plants in jiffy pots grown upon watering for 2 weeks, no watering for 3 weeks and after one week of re-watering. All plots represent the mean of 3 biological replicates (technical replicates n = 36). Whiskers represents minimum and maximum values, "+" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (*** p < 0.001).


J . Confocal microscopy showing the colonization of SA190 : GFP on five days - old seedlings grown on ½ MS and 25 % PEG . Green signal corresponds to GFP - tagged SA190 while in magenta are plant cell wall stained with propidium iodide ( PI ) . Bar indicates 100 nm . K . SA190 colonization levels in Arabidopsis under normal ( ½ MS ) and drought proxy ( ½ MS + 25 % PEG ) conditions as quantified by colony forming units of bacteria per mg of plant fresh weight . Boxplot shows data from four independent biological replicates where whiskers represents minimum and maximum values , " + " indicates mean and horizontal line represents median . Asterisks indicate the statistical differences based on the Student ' s t - test ( * p < 0 . 05 , * * * 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 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 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

J. Confocal microscopy showing the colonization of SA190:GFP on five days-old seedlings grown on ½ MS and 25% PEG. Green signal corresponds to GFP-tagged SA190 while in magenta are plant cell wall stained with propidium iodide (PI). Bar indicates 100 nm. K. SA190 colonization levels in Arabidopsis under normal (½ MS) and drought proxy (½ MS + 25% PEG) conditions as quantified by colony forming units of bacteria per mg of plant fresh weight. Boxplot shows data from four independent biological replicates where whiskers represents minimum and maximum values, "+" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (* p < 0.05, *** p < 0.001).


F - G . Primary root length and Lateral root density of 21 - day - old , mock - and SA190 - colonized Col - 0 , aba2 - 1 and qpyr / pyl plants upon growth for 16 days on ½ MS + 25 % PEG . Data information : Boxplot shows data from three independent biological replicates ( technical replicates n = 36 ) where whiskers represents minimum and maximum values , " + " indicates mean and horizontal line represents median . Asterisks indicate the statistical differences based on the Student ' s t - test ( * p < 0 . 05 , * * * 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

F-G. Primary root length and Lateral root density of 21-day-old, mock- and SA190-colonized Col-0, aba2-1 and qpyr/pyl plants upon growth for 16 days on ½ MS + 25% PEG. Data information: Boxplot shows data from three independent biological replicates (technical replicates n = 36) where whiskers represents minimum and maximum values, "+" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (* p < 0.05, *** p < 0.001).


B . Relative gene expression of aquaporin PIP and TIP genes by qRT - PCR analysis normalized to tubulin levels in 21 - days old mock - and SA190 - colonized roots of Arabidopsis grown for 16 days on ½ MS with or without 25 % PEG . Data information : Values represent the means of three biological experiments . Error bars indicate SE . All plots represent the mean of 3 biological replicates ( biological replicates n = 3 ) . Asterisks indicate a statistical difference based on the Student ' s t - test ( * p < 0 . 05 ; * * 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 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

B. Relative gene expression of aquaporin PIP and TIP genes by qRT-PCR analysis normalized to tubulin levels in 21-days old mock- and SA190-colonized roots of Arabidopsis grown for 16 days on ½ MS with or without 25% PEG. Data information: Values represent the means of three biological experiments. Error bars indicate SE. All plots represent the mean of 3 biological replicates (biological replicates n= 3). Asterisks indicate a statistical difference based on the Student's t-test (* p < 0.05; ** p < 0.01; *** p < 0.001).


C . Relative gene expression of aquaporin PIP and TIP genes by qRT - PCR analysis in Col - 0 , aba2 - 1 and qpyr / pyl mutants normalized to tubulin levels in 21 - days old mock - and SA190 - colonized roots of Arabidopsis grown for 16 days on ½ MS with or without 25 % PEG . Data information : Values represent the means of three biological experiments . Error bars indicate SE . All plots represent the mean of 3 biological replicates ( biological replicates n = 3 ) . Asterisks indicate a statistical difference based on the Student ' s t - test ( * p < 0 . 05 ; * * 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 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

C. Relative gene expression of aquaporin PIP and TIP genes by qRT-PCR analysis in Col-0, aba2-1 and qpyr/pyl mutants normalized to tubulin levels in 21-days old mock- and SA190-colonized roots of Arabidopsis grown for 16 days on ½ MS with or without 25% PEG. Data information: Values represent the means of three biological experiments. Error bars indicate SE. All plots represent the mean of 3 biological replicates (biological replicates n= 3). Asterisks indicate a statistical difference based on the Student's t-test (* p < 0.05; ** p < 0.01; *** p < 0.001).


Relative enrichment of the H3K4me3 mark at the indicated gene loci of selected PIPs and TIPs of non - colonized ( Mock ) and SA190 - colonized ( SA190 ) plants in Col - 0 and aba2 - 1 mutant as determined by chromatin immunoprecipitation - qPCR ( ChIP - qPCR ) . The regions targeted for amplification are labelled as R1 , R2 and R3 . R1 encompasses 5 ' - UTR , R2 is close to the transcription start site ( TSS ) while R3 is outside the gene body . Amplification values were normalized to input and H3 and region 3 ( R3 ) of non - colonized ( Mock ) Col - 0 plants . The plots represent the means of 2 biological replicates . Error bars represent SE . ( ns ) non - significant Asterisks indicate a statistical difference based on 2 - way ANOVA ( * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 for p value differences between the conditions when compared to Col - 0 Mock using Dunnett ' s multiple comparison test ) .

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

Relative enrichment of the H3K4me3 mark at the indicated gene loci of selected PIPs and TIPs of non-colonized (Mock) and SA190-colonized (SA190) plants in Col-0 and aba2-1 mutant as determined by chromatin immunoprecipitation-qPCR (ChIP-qPCR). The regions targeted for amplification are labelled as R1, R2 and R3. R1 encompasses 5'-UTR, R2 is close to the transcription start site (TSS) while R3 is outside the gene body. Amplification values were normalized to input and H3 and region 3 (R3) of non-colonized (Mock) Col-0 plants. The plots represent the means of 2 biological replicates. Error bars represent SE. (ns) non-significant Asterisks indicate a statistical difference based on 2-way ANOVA (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 for p value differences between the conditions when compared to Col-0 Mock using Dunnett's multiple comparison test).


A - B . Total fresh weight of 21 days old non - colonized - ( Mock ) and SA190 - colonized ( SA190 ) Col - 0 , tip2 ; 1 and qpip plants grown for 16 days under normal ( ½ MS ) or drought stress proxy conditions ( ½ MS + 25 % PEG ) . Data information : All plots represent the mean of 3 biological replicates . Error bars represent SE . Boxplot shows data from three independent biological replicates ( technical replicates n = ( A ) 16 - 20 , ( B ) 8 - 20 where whiskers represents minimum and maximum values , " + " indicates mean and horizontal line represents median . Asterisks indicate the statistical differences based on the Student ' s t - test ( * p < 0 . 05 , * * * p < 0 . 001 ) . Asterisks indicate a statistical difference based on the 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 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

A-B. Total fresh weight of 21 days old non-colonized- (Mock) and SA190-colonized (SA190) Col-0, tip2;1 and qpip plants grown for 16 days under normal (½ MS) or drought stress proxy conditions (½ MS + 25% PEG). Data information: All plots represent the mean of 3 biological replicates. Error bars represent SE. Boxplot shows data from three independent biological replicates (technical replicates n = (A) 16-20, (B) 8-20 where whiskers represents minimum and maximum values, "+" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (* p < 0.05, *** p < 0.001). Asterisks indicate a statistical difference based on the Student's t-test *** p < 0.001).


G . Transpiration rate of mock - or SA190 - colonized 21 days old plants after transfer of 5 - day - old seedlings from ½ MS to ½ MS + / - 25 % PEG . Transpiration was quantified as rate of water loss by calculating the changes in fresh weight for 30 min with reading at every 5 min interval .

O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

G. Transpiration rate of mock- or SA190- colonized 21 days old plants after transfer of 5-day-old seedlings from ½ MS to ½ MS +/- 25 % PEG. Transpiration was quantified as rate of water loss by calculating the changes in fresh weight for 30 min with reading at every 5 min interval.


H - I . Comparison of stomatal aperture of SA190 - and non - colonized 21 - days old plants grown for 16 days under non - stress ( ½ MS ) or drought stress proxy ( ½ MS + 25 % PEG ) conditions . Scale bar = 20 µm . Data information : All plots represent the mean of 3 biological replicates . Error bars represent SE . Boxplot shows data from three independent biological replicates ( technical replicates n = ( I ) 94 - 104 , where whiskers represents minimum and maximum values , " + " indicates mean and horizontal line represents median . Asterisks indicate the statistical differences based on the Student ' s t - test ( * p < 0 . 05 , * * * p < 0 . 001 ) . Asterisks indicate a statistical difference based on the 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 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

H-I. Comparison of stomatal aperture of SA190- and non-colonized 21-days old plants grown for 16 days under non-stress (½ MS) or drought stress proxy (½ MS + 25% PEG) conditions. Scale bar = 20 µm. Data information: All plots represent the mean of 3 biological replicates. Error bars represent SE. Boxplot shows data from three independent biological replicates (technical replicates n = (I) 94-104, where whiskers represents minimum and maximum values, "+" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (* p < 0.05, *** p < 0.001). Asterisks indicate a statistical difference based on the Student's t-test *** p < 0.001).


Double immunolabeling of PDS5A ( green ) and SYCP3 ( red ) in mouse spread spermatocytes in

Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in leptotene


Double immunolabeling of PDS5A ( green ) and SYCP3 ( red ) in mouse spread spermatocytes in zygotene Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) 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 O O O O O O O O O O O

Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in zygotene Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Double immunolabeling of PDS5A ( green ) and SYCP3 ( red ) in mouse spread spermatocytes in , early pachytene Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) 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 O O O O O O O O O O O O O

Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in , early pachytene Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Double immunolabeling of PDS5A ( green ) and SYCP3 ( red ) in mouse spread spermatocytes in mid pachytene Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) 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 O O O O O O O O O O O O

Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in mid pachytene Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Double immunolabeling of PDS5A ( green ) and SYCP3 ( red ) in mouse spread spermatocytes in diplotene Arrows in ( I - L ) indicate the position of some chromocenters . Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated . DAPI staining of the chromatin ( blue ) is shown for some spermatocytes .

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

Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in diplotene Arrows in (I-L) indicate the position of some chromocenters. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. DAPI staining of the chromatin (blue) is shown for some spermatocytes.


Double immunolabeling of PDS5A ( green ) and SYCP3 ( red ) in mouse spread spermatocytes in diakinesis Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated .

Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in diakinesis Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


O , P Double immunolabeling of PDS5A ( green ) and kinetochores revealed with an ACA serum ( red ) in a metaphase I spermatocyte . Data information : DAPI staining of the chromatin ( blue ) is shown for some spermatocytes .

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

O, P Double immunolabeling of PDS5A (green) and kinetochores revealed with an ACA serum (red) in a metaphase I spermatocyte. Data information: DAPI staining of the chromatin (blue) is shown for some spermatocytes.


Q , R Double immunolabeling of PDS5A ( green ) and REC8 ( pseudocolored in purple ) in a metaphase I spermatocyte . Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated . DAPI staining of the chromatin ( blue ) is shown for some spermatocytes .

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

Q, R Double immunolabeling of PDS5A (green) and REC8 (pseudocolored in purple) in a metaphase I spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. DAPI staining of the chromatin (blue) is shown for some spermatocytes.


S , T Double immunolabeling of PDS5A ( green ) and kinetochores ( ACA , red ) in a metaphase II spermatocyte . Data information : DAPI staining of the chromatin ( blue ) is shown for some spermatocytes .

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

S, T Double immunolabeling of PDS5A (green) and kinetochores (ACA, red) in a metaphase II spermatocyte. Data information: DAPI staining of the chromatin (blue) is shown for some spermatocytes.


Double immunolabeling of PDS5B ( green ) and SYCP3 ( red ) in spread spermatocytes at the indicated stages .

Double immunolabeling of PDS5B (green) and SYCP3 (red) in spread spermatocytes at the indicated stages.


Double immunolabeling of PDS5B ( green ) and SYCP3 ( red ) in spread spermatocytes at the indicated stages . Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated .

Double immunolabeling of PDS5B (green) and SYCP3 (red) in spread spermatocytes at the indicated stages. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Immunolabeling of PDS5B ( green ) and REC8 ( pseudocolored in blue onto a spread pachytene spermatocyte . Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated .

Immunolabeling of PDS5B (green) and REC8 (pseudocolored in blue onto a spread pachytene spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Immunolabeling of PDS5B ( green ) and SYCP3 ( red ) and TRF1 ( pseudocolored in yellow onto a spread pachytene spermatocyte . Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) 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 O O O O O O O O O O O O O O O O

Immunolabeling of PDS5B (green) and SYCP3 (red) and TRF1 (pseudocolored in yellow onto a spread pachytene spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Immunolabeling of PDS5B ( green ) and ACA ( pseudocolored in purple onto a spread pachytene spermatocyte . Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated .

Immunolabeling of PDS5B (green) and ACA (pseudocolored in purple onto a spread pachytene spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Double immunolabeling of PDS5B ( green ) and SYCP3 ( red ) on diplotene spread nuclei . Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) 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 O O O O O O O O O O

Double immunolabeling of PDS5B (green) and SYCP3 (red) on diplotene spread nuclei. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Double immunolabeling of PDS5B ( green ) and SYCP3 ( red ) on diakinesis spread nuclei . Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated .

Double immunolabeling of PDS5B (green) and SYCP3 (red) on diakinesis spread nuclei. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


A - F Double immunolabeling of PDS5B ( green ) and SYCP3 ( red ) in a metaphase I spermatocyte ( A - B ) and selected bivalents ( C - F ) . The sex bivalent ( XY ) is indicated .

A-F Double immunolabeling of PDS5B (green) and SYCP3 (red) in a metaphase I spermatocyte (A-B) and selected bivalents (C-F). The sex bivalent (XY) is indicated.


G - J Double immunolabeling of PDS5B ( green ) and kinetochores ( ACA , red ) , and staining of the chromatin ( DAPI ) in a metaphase II spermatocyte .

O O O O O O O O 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-J Double immunolabeling of PDS5B (green) and kinetochores (ACA, red), and staining of the chromatin (DAPI) in a metaphase II spermatocyte.


Double immunolabeling of PDS5A ( green , A - E ) and SYCP3 ( red ) in Pds5AB cKO spread spermatocytes at the indicated stages .

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

Double immunolabeling of PDS5A (green, A-E) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages.


Double immunolabeling of PDS5B ( green , F - J ) and SYCP3 ( red ) in Pds5AB cKO spread spermatocytes at the indicated stages .

Double immunolabeling of PDS5B (green, F-J) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages.


K - M Preparations from the same mice were immunolabeled with the testis - specific histone variant H1t ( green ) and SYCP3 ( red ) .

O O O 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-M Preparations from the same mice were immunolabeled with the testis-specific histone variant H1t (green) and SYCP3 (red).


N Scatter dot - plot graph of total SC length measured in the autosomal pairs of 33 early pachytene cells from three different individuals of each genotype . Bars indicate mean and standard error . Statistical significance was assessed using an unpaired two - tailed T - test function ( * * * * , 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

N Scatter dot-plot graph of total SC length measured in the autosomal pairs of 33 early pachytene cells from three different individuals of each genotype. Bars indicate mean and standard error. Statistical significance was assessed using an unpaired two-tailed T-test function (****, p < 0.0001).


A - H Double immunolabeling of SYCP1 ( green ) and SYCP3 ( red ) in wild - type ( A - D ) and Pds5AB cKO ( E - H ) spread spermatocytes at the indicated stages . I - L Double immunolabeling of HORMAD2 ( pseudocolored in white ) and SYCP3 ( red ) . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 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

A-H Double immunolabeling of SYCP1 (green) and SYCP3 (red) in wild-type (A-D) and Pds5AB cKO (E-H) spread spermatocytes at the indicated stages. I-L Double immunolabeling of HORMAD2 (pseudocolored in white) and SYCP3 (red). Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and the cohesin subunits SMC3 ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 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

Double immunolabeling of SYCP3 (red) and the cohesin subunits SMC3 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and the cohesin subunits REC8 ( green in Pds5AB cKO spread spermatocytes at the indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and the cohesin subunits REC8 (green in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and the cohesin subunits SMC1β ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and the cohesin subunits SMC1β (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and the cohesin cofactors Sororin ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and the cohesin cofactors Sororin (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and the cohesin cofactors WAPL ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and the cohesin cofactors WAPL (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and either γH2AX ( pseudocolored in blue in Pds5AB cKO spread spermatocytes at the indicated stages .

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

Double immunolabeling of SYCP3 (red) and either γH2AX (pseudocolored in blue in Pds5AB cKO spread spermatocytes at the indicated stages.


Double immunolabeling of SYCP3 ( red ) and RAD51 ( green in Pds5AB cKO spread spermatocytes at the indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and RAD51 (green in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


I Scatter dot - plot graph of the quantification of RAD51 foci in in 15 zygotene nuclei randomly selected from three different individuals of each genotype . Bars indicate mean and standard error and statistical significance was assessed with an unpaired two - tailed T - test function ( * * * * , 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

I Scatter dot-plot graph of the quantification of RAD51 foci in in 15 zygotene nuclei randomly selected from three different individuals of each genotype. Bars indicate mean and standard error and statistical significance was assessed with an unpaired two-tailed T-test function (****, p < 0.0001).


J - L Double immunolabeling of MLH1 ( pseudocolored in white ) and SYCP3 ( red ) in Pds5AB cKO spread spermatocytes . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

J-L Double immunolabeling of MLH1 (pseudocolored in white) and SYCP3 (red) in Pds5AB cKO spread spermatocytes. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and TRF1 ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and TRF1 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and RAP1 ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and RAP1 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


I Selected zygotene - like and pachytene - like bivalents presenting altered TRF1 distribution as ( from left to right ) ( i ) telomere stretches , ( ii ) multiple telomere , ( iii ) distant telomeres and ( iv ) telomere - less . Arrowheads in ( I ) indicate the position of altered telomeres . J Quantification of telomeres with a regular or altered disposition of TRF1 in Pds5AB cKO spermatocytes at the indicated stages ( n = 1646 telomeres in zygotene - like spermatocytes and 738 in pachytene - like ones ) . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Selected zygotene-like and pachytene-like bivalents presenting altered TRF1 distribution as (from left to right) (i) telomere stretches, (ii) multiple telomere, (iii) distant telomeres and (iv) telomere-less. Arrowheads in (I) indicate the position of altered telomeres. J Quantification of telomeres with a regular or altered disposition of TRF1 in Pds5AB cKO spermatocytes at the indicated stages (n=1646 telomeres in zygotene-like spermatocytes and 738 in pachytene-like ones). Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


K , L As in ( I , J ) , respectively , but for RAP1 staining ( n = 1376 telomeres in zygotene - like spermatocytes and 328 in pachytene - like ones ) . Arrowheads in ( K ) indicate the position of altered telomeres . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 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

K, L As in (I, J), respectively, but for RAP1 staining (n=1376 telomeres in zygotene-like spermatocytes and 328 in pachytene-like ones). Arrowheads in (K) indicate the position of altered telomeres. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


M - P Double immunolabeling of SUN1 ( green ) and SYCP3 ( red ) in Pds5AB cKO spread spermatocytes at the indicated stages . 0 , 1 and 2 in ( N and O ) indicate the number of chromosome ends labelled by SUN1 in a given bivalent . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 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

M-P Double immunolabeling of SUN1 (green) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages. 0, 1 and 2 in (N and O) indicate the number of chromosome ends labelled by SUN1 in a given bivalent. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Q As in ( I ) , but for SUN1 staining . Arrowheads in ( Q ) indicate the position of altered telomeres . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Q As in (I), but for SUN1 staining. Arrowheads in (Q) indicate the position of altered telomeres. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


R Selected zygotene - like and early pachytene - like bivalents from Pds5AB cKO spermatocytes immunolabeled with SYCP3 ( red ) and SUN1 ( green ) , and their respective percentage of appearance . S Quantification of telomeres with a regular or altered disposition of SUN1 ( n = 715 telomeres in zygotene - like spermatocytes and 410 in pachytene - like ones ) . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 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 O O O O O

R Selected zygotene-like and early pachytene-like bivalents from Pds5AB cKO spermatocytes immunolabeled with SYCP3 (red) and SUN1 (green), and their respective percentage of appearance. S Quantification of telomeres with a regular or altered disposition of SUN1 (n=715 telomeres in zygotene-like spermatocytes and 410 in pachytene-like ones). Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


A - C Immunolabeling of SYCP3 ( red ) and telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytes at the indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 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

A-C Immunolabeling of SYCP3 (red) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


D 300 % magnification of selected zygotene - like and pachytene - like bivalents presenting regular ( left ) or altered ( i - iv ) telomere FISH signals .

O O O O O O 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 300% magnification of selected zygotene-like and pachytene-like bivalents presenting regular (left) or altered (i-iv) telomere FISH signals.


Double immunolabeling of SYCP3 ( red ) and RAP1 ( blue ) and telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread

Double immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes


Double immunolabeling of SYCP3 ( red ) and RAP1 ( blue ) and telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytes at the indicated stages . Asterisks indicate the position of the enlarged chromosomes / bivalents shown in a 300 % magnification displaying telomere stretches , multiple telomere , distant telomeres and telomere - less . White arrowheads indicate telomeres with an altered organization of telomeric DNA into multiple telomere signals with RAP1 signals and telomere stretches Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 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 O O O O O O O O O O O O O O O O O O O O O O O

Double immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages. Asterisks indicate the position of the enlarged chromosomes/bivalents shown in a 300% magnification displaying telomere stretches, multiple telomere, distant telomeres and telomere-less. White arrowheads indicate telomeres with an altered organization of telomeric DNA into multiple telomere signals with RAP1 signals and telomere stretches Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and RAP1 ( blue ) and telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytes at the indicated stages . Asterisks indicate the position of the enlarged chromosomes / bivalents shown in a 300 % magnification displaying telomere stretches , multiple telomere , distant telomeres and telomere - less . White arrowheads indicate telomeres with an altered organization of telomeric DNA into multiple telomere signals without RAP1 signals telomere stretches Pink arrowheads in ( T ) indicate a telomere presenting a regular telomere FISH signal without a RAP1 signal . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Double immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages. Asterisks indicate the position of the enlarged chromosomes/bivalents shown in a 300% magnification displaying telomere stretches, multiple telomere, distant telomeres and telomere-less. White arrowheads indicate telomeres with an altered organization of telomeric DNA into multiple telomere signals without RAP1 signals telomere stretches Pink arrowheads in (T) indicate a telomere presenting a regular telomere FISH signal without a RAP1 signal. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) and RAP1 ( blue ) and telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytes at the indicated stages . Asterisks indicate the position of the enlarged chromosomes / bivalents shown in a 300 % magnification displaying telomere stretches , multiple telomere , distant telomeres and telomere - less .

Double immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages. Asterisks indicate the position of the enlarged chromosomes/bivalents shown in a 300% magnification displaying telomere stretches, multiple telomere, distant telomeres and telomere-less.


Double immunolabeling of TRF1 ( green ) and SYCP3 ( red ) in squashed spermatocytes from wild - type or Pds5AB cKO mice M - P ) . The first three columns correspond to z - projections of 15 focal planes throughout the Top , Equator and Bottom regions of the nucleus . The projection of 65 focal planes across the same spermatocyte nucleus is shown at the fourth column ( Z - Projection ) . White arrowheads in ( F indicate the presence of TRF1 signals non - attached to the NE . Scale bar , 10 μm .

Double immunolabeling of TRF1 (green) and SYCP3 (red) in squashed spermatocytes from wild-type or Pds5AB cKO mice M-P). The first three columns correspond to z-projections of 15 focal planes throughout the Top, Equator and Bottom regions of the nucleus. The projection of 65 focal planes across the same spermatocyte nucleus is shown at the fourth column (Z-Projection). White arrowheads in (F indicate the presence of TRF1 signals non-attached to the NE. Scale bar, 10 μm.


Double immunolabeling of TRF1 ( green ) and SYCP3 ( red ) in squashed spermatocytes from wild - type or Pds5AB cKO mice The first three columns correspond to z - projections of 15 focal planes throughout the Top , Equator and Bottom regions of the nucleus . The projection of 65 focal planes across the same spermatocyte nucleus is shown at the fourth column ( Z - Projection ) . White arrowheads in N ) indicate the presence of TRF1 signals non - attached to the NE . Scale bar , 10 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 O O O O O O O O O O O O O O O O O O

Double immunolabeling of TRF1 (green) and SYCP3 (red) in squashed spermatocytes from wild-type or Pds5AB cKO mice The first three columns correspond to z-projections of 15 focal planes throughout the Top, Equator and Bottom regions of the nucleus. The projection of 65 focal planes across the same spermatocyte nucleus is shown at the fourth column (Z-Projection). White arrowheads in N) indicate the presence of TRF1 signals non-attached to the NE. Scale bar, 10 μm.


Q - T Selected bivalents from Pds5AB cKO spermatocytes displaying structural telomere abnormalities as telomere stretches ( Q ) , multiple telomere ( R ) , distant telomere ( S ) and telomere - less ( T ) .

Q-T Selected bivalents from Pds5AB cKO spermatocytes displaying structural telomere abnormalities as telomere stretches (Q), multiple telomere (R), distant telomere (S) and telomere-less (T).


D . Immunoblot analysis of caspase - 1 ( Casp - 1 ) , caspase - 11 ( Casp - 11 ) , gasdermin D ( GSDMD ) and GAPDH ( loading control ) in WT and mutant BMDMs left untreated or assessed 20 h after infection with M . catarrhalis ( Ne11 , MOI 100 ) . Data information : Data are from one 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

D. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11), gasdermin D (GSDMD) and GAPDH (loading control) in WT and mutant BMDMs left untreated or assessed 20 h after infection with M. catarrhalis (Ne11, MOI 100). Data information: Data are from one three independent experiments (D


E . Release of IL - 1β , IL - 18 , TNF and LDH from BMDMs after treatment as in D . Data information : Each symbol represents an independent biological replicate ( E ) . * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons test ( E ) . Data are from three independent experiments E ; mean and s . e . m . in

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Release of IL-1β, IL-18, TNF and LDH from BMDMs after treatment as in D. Data information: Each symbol represents an independent biological replicate (E). **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (E). Data are from three independent experiments E; mean and s.e.m. in E


G . IncuCyte live - imaging analysis of WT and Casp11 - / - BMDM viability after infection with M . catarrhalis as in D , or transfection with LPS . Data information : Data are pooled from two ( G ) independent experiments mean and s . e . m . in G ) .

O O O O O 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

G. IncuCyte live-imaging analysis of WT and Casp11-/- BMDM viability after infection with M. catarrhalis as in D, or transfection with LPS. Data information: Data are pooled from two (G) independent experiments mean and s.e.m. in G).


A . Immunoblot analysis of caspase - 1 ( Casp - 1 ) , caspase - 11 ( Casp - 11 ) and gasdermin D ( GSDMD ) in WT , Nlrp3 - / - , Casp11 - / - and Aim2 - / - BMDMs left untreated ( Med . ) or 5 h after transfection with 5 µg of LOS from M . catarrhalis ( O35E ) or 5 µg of LPS from E . coli . Data information : Data are from three independent experiments ( 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 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

A. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11) and gasdermin D (GSDMD) in WT, Nlrp3-/-, Casp11-/- and Aim2-/- BMDMs left untreated (Med.) or 5 h after transfection with 5 µg of LOS from M. catarrhalis (O35E) or 5 µg of LPS from E. coli. Data information: Data are from three independent experiments (A,


B . Release of IL - 1β , IL - 18 , TNF and LDH from BMDMs after treatment as in A . Data information : Each symbol represents an independent biological replicate ( B , NS , no statistical significance ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons test ( B , Data are pooled from three independent experiments ( B , mean and s . e . m . in B ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Release of IL-1β, IL-18, TNF and LDH from BMDMs after treatment as in A. Data information: Each symbol represents an independent biological replicate (B, NS, no statistical significance; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (B, Data are pooled from three independent experiments (B, mean and s.e.m. in B,


F . Transcript abundance for genes encoding GTPases in WT and Ifnar1 - / - BMDMs infected with M . catarrhalis as in C . Transcript abundance is provided in counts per million ( CPM ) . Data information : Data are pooled from three independent

F. Transcript abundance for genes encoding GTPases in WT and Ifnar1-/- BMDMs infected with M. catarrhalis as in C. Transcript abundance is provided in counts per million (CPM). Data information: Data are pooled from three independent experiments


G . qRT - PCR analysis of genes encoding the family of guanylate binding proteins ( GBP1 - 11 ) in WT and Ifnar1 - / - BMDMs left untreated ( Med . ) or following treated as in C . Data information : Each symbol represents an independent biological replicate G ) . NS , no statistical significance ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons test G ) ) . Data are from one experiment representative of four ( G ) independent experiments ( mean and s . e . m . in G ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. qRT-PCR analysis of genes encoding the family of guanylate binding proteins (GBP1-11) in WT and Ifnar1-/- BMDMs left untreated (Med.) or following treated as in C. Data information: Each symbol represents an independent biological replicate G). NS, no statistical significance; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test G)). Data are from one experiment representative of four (G) independent experiments (mean and s.e.m. in G).


A . Confocal microscopy analysis of intracellular M . catarrhalis ( green ) and GBP1 , GBP2 , GBP3 , GBP5 or GBP7 ( red ) in WT BMDMs left untreated ( Med . ) or assessed 12 h after infection with M . catarrhalis ( Ne11 , MOI 20 ) . B . Quantitation of GBP1 - , GBP2 - , GBP3 - , GBP5 - and GBP7 - postive M . catarrhalis in WT BMDMs as treated in A . Data information : Arrowheads indicate bacteria colocalised with GBPs . To quantify the proportion of GBP - positive bacteria in a field of view , we used the single red channel ( anti - GBP staining ) and compared this to the green channel ( anti - M . catarrhalis staining ) . The number of GBP - positive M . catarrhalis was divided by the total number of M . catarrhalis counted to obtain the proportion of GBP - positive bacteria ( B ) . A total of 100 BMDMs were analysed to quantify the proportion of GBP - positive bacteria per cell using confocal microscopy ( B ) Data are from three independent experiments ( A , or are pooled from two ( B ) independent experiments Scale bars , 5 µm ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 analysis of intracellular M. catarrhalis (green) and GBP1, GBP2, GBP3, GBP5 or GBP7 (red) in WT BMDMs left untreated (Med.) or assessed 12 h after infection with M. catarrhalis (Ne11, MOI 20). B. Quantitation of GBP1-, GBP2-, GBP3-, GBP5- and GBP7-postive M. catarrhalis in WT BMDMs as treated in A. Data information: Arrowheads indicate bacteria colocalised with GBPs. To quantify the proportion of GBP-positive bacteria in a field of view, we used the single red channel (anti-GBP staining) and compared this to the green channel (anti-M. catarrhalis staining). The number of GBP-positive M. catarrhalis was divided by the total number of M. catarrhalis counted to obtain the proportion of GBP-positive bacteria (B). A total of 100 BMDMs were analysed to quantify the proportion of GBP-positive bacteria per cell using confocal microscopy (B) Data are from three independent experiments (A, or are pooled from two (B) independent experiments Scale bars, 5 µm (A,


F . Recovery of M . catarrhalis ( as colony - forming units ( CFU ) from primary WT , Gbpchr3 - KO and Ifnar1 - / - lung fibroblasts 4 , 8 and 12 h after infection with M . catarrhalis ( Ne11 , MOI 100 ) . Data information : Each symbol represents an independent biological replicate F ) . NS , no statistical significance ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons test Data are from three independent experiments F ; mean and 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

F. Recovery of M. catarrhalis (as colony-forming units (CFU) from primary WT, Gbpchr3-KO and Ifnar1-/- lung fibroblasts 4, 8 and 12 h after infection with M. catarrhalis (Ne11, MOI 100). Data information: Each symbol represents an independent biological replicate F). NS, no statistical significance; * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test Data are from three independent experiments F; mean and s.e.m.


G . Quantitation of intracellular bacteria by Transmission electron microscopy ( TEM ) in WT , Gbpchr3 - KO and Ifnar1 - / - BMDMs infected with M . catarrhalis ( Ne11 , MOI 50 ) for 12 h . Each symbol represents an independent experiment ( E and F ) . Data information : A total of 100 BMDMs were analysed to quantify the intracellular bacterial burden using electron microscopy ( G ) . Data are from one experiment representative of two ( G ) independent experiments mean and 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

G. Quantitation of intracellular bacteria by Transmission electron microscopy (TEM) in WT, Gbpchr3-KO and Ifnar1-/- BMDMs infected with M. catarrhalis (Ne11, MOI 50) for 12 h. Each symbol represents an independent experiment (E and F). Data information: A total of 100 BMDMs were analysed to quantify the intracellular bacterial burden using electron microscopy (G). Data are from one experiment representative of two (G) independent experiments mean and s.e.m.


A . Immunoblot analysis of caspase - 1 ( Casp - 1 ) , caspase - 11 ( Casp - 11 ) and gasdermin D ( GSDMD ) in WT , Gbp1 - / - , Gbp2 - / - , Gbp3 - / - , Gbp5 - / - , Gbp7 - / - , Gbp4 / 8 / 9 - / - , Gbp11 - / - and Casp11 - / - BMDMs left untreated ( Med . ) or assessed 10 h after infection with M . catarrhalis ( Ne11 , MOI 100 ) , or 5 h after transfection with 5 µg LPS from 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

A. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11) and gasdermin D (GSDMD) in WT, Gbp1-/-, Gbp2-/-, Gbp3-/-, Gbp5-/-, Gbp7-/-, Gbp4/8/9-/-, Gbp11-/- and Casp11-/- BMDMs left untreated (Med.) or assessed 10 h after infection with M. catarrhalis (Ne11, MOI 100), or 5 h after transfection with 5 µg LPS from E. coli.


B . Release of IL - 1β , IL - 18 , LDH , TNF , IL - 6 and KC from BMDMs after treatment as in A . Data information : Each symbol represents an independent biological replicate ( B ) . NS , no statistical significance ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons test ( B ) ) . Data are are pooled from three independent experiments ( B ; mean and s . e . m . in B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Release of IL-1β, IL-18, LDH, TNF, IL-6 and KC from BMDMs after treatment as in A. Data information: Each symbol represents an independent biological replicate (B). NS, no statistical significance; * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (B)). Data are are pooled from three independent experiments (B; mean and s.e.m. in B).


B . Quantification of OD600 of M . catarrhalis in BHI media over 6 h in the presence of solvent control ( Sol . Ctrl . ) , 80 µg / mL recombinant GBP2 or 50 µg / mL kanamycin . Data information : * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons test ( B Data are from one experiment representative of two ( A ) or three ( B , Each independent experiment consists of three ( B , technical 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 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

B. Quantification of OD600 of M. catarrhalis in BHI media over 6 h in the presence of solvent control (Sol. Ctrl.), 80 µg/mL recombinant GBP2 or 50 µg/mL kanamycin. Data information: ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (B Data are from one experiment representative of two (A) or three (B, Each independent experiment consists of three (B, technical replicates.


C . Quantification of ATP from M . catarrhalis in BHI media over 6 h in the presence of Sol . Ctrl . , 30 µg / mL recombinant GBP2 or 50 µg / mL kanamycin . Data information : Data are pooled from two ( C ) independent experiments Each independent experiment consists of two ( C ) technical replicates .

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 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

C. Quantification of ATP from M. catarrhalis in BHI media over 6 h in the presence of Sol. Ctrl., 30 µg/mL recombinant GBP2 or 50 µg/mL kanamycin. Data information: Data are pooled from two (C) independent experiments Each independent experiment consists of two (C) technical replicates.


D . Viability of M . catarrhalis ( as a percentage of CFU in relation to Sol . Ctrl . ) assessed 6 h after incubation with recombinant GBP2 at 3 , 30 or 300 µg / mL . Data information : * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons test D ) Data are from one experiment representative of three D , independent experiments Each independent experiment consists of three D , technical 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Viability of M. catarrhalis (as a percentage of CFU in relation to Sol. Ctrl.) assessed 6 h after incubation with recombinant GBP2 at 3, 30 or 300 µg/mL. Data information: ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test D) Data are from one experiment representative of three D, independent experiments Each independent experiment consists of three D, technical replicates.


A , B . Bacterial burden and concentration of IL - 18 in the spleen of WT mice ( n = 10 ) and Casp11 - / - mice ( n = 10 ) after M . catarrhalis infection . C , D . Bacterial burden and concentration of IL - 18 in the spleen of WT mice ( n = 10 ) and Nlrp3 - / - mice ( n = 11 ) after M . catarrhalis infection . E , F . Bacterial burden and concentration of IL - 18 in the spleen of WT mice ( n = 10 ) and Gbp1 - / - mice ( n = 9 ) after M . catarrhalis infection . G , H . Bacterial burden and concentration of IL - 18 in the spleen of WT mice ( n = 10 ) and Gbp2 - / - mice ( n = 8 ) after M . catarrhalis infection . I , J . Bacterial burden and concentration of IL - 18 in the spleen of WT mice ( n = 10 ) and Gbp3 - / - mice ( n = 8 ) after M . catarrhalis infection . K , L . Bacterial burden and concentration of IL - 18 in the spleen of WT mice ( n = 5 ) and Gbp5 - / - mice ( n = 8 ) after M . catarrhalis infection . Data information : A mix of male and female mice 6 - 8 weeks old were used in each experiment and were injected i . p . with 2 × 107 CFU of M . catarrhalis and analysed after 6 h . Each symbol represents an individual mouse ( A - L ) . Each panel represents data from a single experiment ( mean and s . e . m . in A - L ) . Each experiment was performed at least two times . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( two - tailed t - test ( A - L ) ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Casp11-/- mice (n=10) after M. catarrhalis infection. C, D. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Nlrp3-/- mice (n=11) after M. catarrhalis infection. E, F. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Gbp1-/- mice (n=9) after M. catarrhalis infection. G, H. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Gbp2-/- mice (n=8) after M. catarrhalis infection. I, J. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Gbp3-/- mice (n=8) after M. catarrhalis infection. K, L. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=5) and Gbp5-/- mice (n=8) after M. catarrhalis infection. Data information: A mix of male and female mice 6-8 weeks old were used in each experiment and were injected i.p. with 2×107 CFU of M. catarrhalis and analysed after 6 h. Each symbol represents an individual mouse (A-L). Each panel represents data from a single experiment (mean and s.e.m. in A-L). Each experiment was performed at least two times. * P<0.05; ** P<0.01; *** P<0.001 (two-tailed t-test (A-L)).


( D ) At 48h after transfection of H7 plasmid , the transfectants were incubated with TPCK - trypsin in the presence of recombinant wtSPINK6 protein or PBS or a protease inhibitor AEBSF for 30 min and then applied to examine HA cleavage .

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

(D) At 48h after transfection of H7 plasmid, the transfectants were incubated with TPCK-trypsin in the presence of recombinant wtSPINK6 protein or PBS or a protease inhibitor AEBSF for 30 min and then applied to examine HA cleavage.


( E ) At 24h after transfection of wtSPINK6 plasmid or blank vector in triplicate , A549 cells were inoculated with H7N9 / ah virus . At the indicated hours post infection ( hpi ) , culture media ( supernatant ) and cell lysate were harvested for viral load detection . Data are presented as mean and SD in a representative experiment performed 3 times . * * , P < 0 . 01 ; * * * , P < 0 . 001 . Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) At 24h after transfection of wtSPINK6 plasmid or blank vector in triplicate, A549 cells were inoculated with H7N9/ah virus. At the indicated hours post infection (hpi), culture media (supernatant) and cell lysate were harvested for viral load detection. Data are presented as mean and SD in a representative experiment performed 3 times. **, P<0.01; ***, P<0.001. Student's t test.


( A ) At 48 hours post transfection of the indicated protease plasmids in triplicate , BHK21 cells were incubated with a fluorogenic substrate and wtSPINK6 or loss - of - function mutSPINK6 or PBS for 2 hours and then applied to fluorescence assay . Data represent mean and SD of triplicated wells in a representative experiment performed 3 times . * * , P < 0 . 01 . Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) At 48 hours post transfection of the indicated protease plasmids in triplicate, BHK21 cells were incubated with a fluorogenic substrate and wtSPINK6 or loss-of-function mutSPINK6 or PBS for 2 hours and then applied to fluorescence assay. Data represent mean and SD of triplicated wells in a representative experiment performed 3 times. **, P<0.01. Student's t test.


( B ) At 48h after co - transfection of the indicated protease plasmids and H7 or H1 plasmid , BHK21 cells were lysed to examine HA cleavage with WB .

O O O O O O O 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) At 48h after co-transfection of the indicated protease plasmids and H7 or H1 plasmid, BHK21 cells were lysed to examine HA cleavage with WB.


( E ) At 48h after triple - transfection of H1 , the indicated proteases and wtSPINK6 or mutSPINK6 , BHK21 cells were lysed for the detection of HA cleavage .

(E) At 48h after triple-transfection of H1, the indicated proteases and wtSPINK6 or mutSPINK6, BHK21 cells were lysed for the detection of HA cleavage.


( A ) At 36h after transfection of the indicted protease and wtSPINK6 plasmids or blank vector in triplicate , BHK21 cells were inoculated with H7N9 / ah at a MOI of 0 . 5 . Cell - free culture media were harvested at 24 hpi for viral titration . Data represent the mean and SD of the triplicated wells in a representative experiment performed 3 times . * * , P < 0 . 01 . Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) At 36h after transfection of the indicted protease and wtSPINK6 plasmids or blank vector in triplicate, BHK21 cells were inoculated with H7N9/ah at a MOI of 0.5. Cell-free culture media were harvested at 24 hpi for viral titration. Data represent the mean and SD of the triplicated wells in a representative experiment performed 3 times. **, P<0.01. Student's t test.


( B ) At 24h after transfection of HAT or KLK5 plasmid in sextuplicate , A549 cells were inoculated with H1N1 / pdm at MOI of 0 . 25 . The infected cells were incubated with recombinant wtSPINK6 or mutSPINK6 protein in triplicate for 24h . Culture media in each well were stored in two aliquots , one was applied to viral titration with conventional plaque assay ( black bars ) , the other was pre - treated with TPCK - trypsin for 1 hour prior to plaque assay ( grey bars ) . Data represent the mean and SD of the triplicated wells in a representative experiment performed 3 times . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) At 24h after transfection of HAT or KLK5 plasmid in sextuplicate, A549 cells were inoculated with H1N1/pdm at MOI of 0.25. The infected cells were incubated with recombinant wtSPINK6 or mutSPINK6 protein in triplicate for 24h. Culture media in each well were stored in two aliquots, one was applied to viral titration with conventional plaque assay (black bars), the other was pre-treated with TPCK-trypsin for 1 hour prior to plaque assay (grey bars). Data represent the mean and SD of the triplicated wells in a representative experiment performed 3 times. *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.


( A ) Confocal imaging ( left ) shows intrinsic SPINK6 ( green ) expression in the epithelial cells in 2D human airway organoids . Nuclei and actin filaments are counterstained and shown as blue and purple respectively . Scale , 10 µm . Flow cytometry analysis demonstrates HAT and SPINK6 expression in human airway organoids . The plots ( middle ) show the representative result of one experiment performed 3 times . The percentages of HAT and SPINK6 positive cells ( right ) are shown as mean and SD of 3 independent experiments . Data information : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 imaging (left) shows intrinsic SPINK6 (green) expression in the epithelial cells in 2D human airway organoids. Nuclei and actin filaments are counterstained and shown as blue and purple respectively. Scale, 10 µm. Flow cytometry analysis demonstrates HAT and SPINK6 expression in human airway organoids. The plots (middle) show the representative result of one experiment performed 3 times. The percentages of HAT and SPINK6 positive cells (right) are shown as mean and SD of 3 independent experiments. Data information: *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.