( C ) Primary hepatocytes prepared from p62f / f ; Alb - Cre mice were co - infected with adenovirus GFP or NBR1 in combination with wild - type p62 or the mutants for 48 hrs . Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies . Data shown are representative of three separate experiments . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin . Data are shown as means ± s . e . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 as determined by Welch ' 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

(C) Primary hepatocytes prepared from p62f/f; Alb-Cre mice were co-infected with adenovirus GFP or NBR1 in combination with wild-type p62 or the mutants for 48 hrs. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05, **P < 0.01, and ***P < 0.001 as determined by Welch's t-test.


( D ) RT qPCR analysis . Total RNAs were prepared from hepatocytes described in ( C ) . Values were normalized against the amount of mRNA in the hepatocytes expressing GFP . The experiments were performed three times . Data are shown as means ± s . e . * P < 0 . 05 , and * * P < 0 . 01 as determined by Welch ' 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

(D) RT qPCR analysis. Total RNAs were prepared from hepatocytes described in (C). Values were normalized against the amount of mRNA in the hepatocytes expressing GFP. The experiments were performed three times. Data are shown as means ± s.e. *P < 0.05, and **P < 0.01 as determined by Welch's t-test.


( A ) Immunoblot analysis . Primary hepatocytes prepared from Nrf2f / f and Nrf2f / f ; Alb - Cre mice were infected with adenovirus expressing GFP or NBR1 for 48 hrs . Cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis with the indicated antibodies . Data shown are representative of three separate experiments . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin ( cytosolic proteins ) or Lamin B ( nuclear proteins ) . Data are shown as means ± s . e . * P < 0 . 05 , and * * P < 0 . 01 as determined by Welch ' s t - test .

(A) Immunoblot analysis. Primary hepatocytes prepared from Nrf2f/f and Nrf2f/f; Alb-Cre mice were infected with adenovirus expressing GFP or NBR1 for 48 hrs. Cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin (cytosolic proteins) or Lamin B (nuclear proteins). Data are shown as means ± s.e. *P < 0.05, and **P < 0.01 as determined by Welch's t-test.


( B ) Real - time PCR . Total RNAs were prepared from primary hepatocytes described in ( A ) . Values were normalized against the amount of mRNA in the Nrf2f / f hepatocytes expressing GFP . The experiments were performed at least three times . Data are shown as means ± s . e . * P < 0 . 05 as determined by Welch ' 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

(B) Real-time PCR. Total RNAs were prepared from primary hepatocytes described in (A). Values were normalized against the amount of mRNA in the Nrf2f/f hepatocytes expressing GFP. The experiments were performed at least three times. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.


( C ) Immunofluorescence microscopy . Hepatocytes isolated from Nrf2f / f and Nrf2f / f ; Alb - Cre mice were infected with LacZ or NBR1 adenovirus for 48 hrs and then immunostained with anti - p62 and anti - NBR1 antibodies . Each inset is a magnified image . Bars : 20 µm . The number and size of p62 - bodies were measured in more than 100 cells . Data are shown as means ± s . e . * * * P < 0 . 001 as determined by Welch ' 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

(C) Immunofluorescence microscopy. Hepatocytes isolated from Nrf2f/f and Nrf2f/f; Alb-Cre mice were infected with LacZ or NBR1 adenovirus for 48 hrs and then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. ***P < 0.001 as determined by Welch's t-test.


( A ) Immunoblot analysis . Primary hepatocytes prepared from wild - type mice were infected with the indicated adenovirus for 48 hrs and subsequently treated with 100 nM Bafilomycin A1 ( BafA1 ) for 24 hrs or cultured in amino acid - deprived medium ( Stv ) for 12 hrs . Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies . Data shown are representative of three separate experiments . Bar graphs show the quantitative densitometric analysis of the indicated proteins relative to actin . Data are shown as means ± s . e . * P < 0 . 05 as determined by Welch ' 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 B-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O

(A) Immunoblot analysis. Primary hepatocytes prepared from wild-type mice were infected with the indicated adenovirus for 48 hrs and subsequently treated with 100 nM Bafilomycin A1 (BafA1) for 24 hrs or cultured in amino acid-deprived medium (Stv) for 12 hrs. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs show the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.


( B ) Fluorescence microscopy . p62 - deficient MEFs were co - transfected with mCherry - GFP - p62 in combination with wild - type NBR1 or NBR1 D50R mutant . 48 hrs after the transfection , fluorescence images were observed . Bars : 10 µm . The number of puncta positive for only red - signal in each MEFs was counted in more than 100 cells . Bar graphs are represented as means ± s . e . * * * P < 0 . 001 as determined by Welch ' 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

(B) Fluorescence microscopy. p62-deficient MEFs were co-transfected with mCherry-GFP-p62 in combination with wild-type NBR1 or NBR1 D50R mutant. 48 hrs after the transfection, fluorescence images were observed. Bars: 10 µm. The number of puncta positive for only red-signal in each MEFs was counted in more than 100 cells. Bar graphs are represented as means ± s.e. ***P < 0.001 as determined by Welch's t-test.


( A ) Immunoblot analysis . Primary hepatocytes were isolated from wild - type ( wt ) , p62f / f ; Alb - Cre ( p62KO ) and Nrf2f / f ; Alb - Cre ( Nrf2KO ) mice . The hepatocytes were challenged with 10 μM sodium arsenite [ As ( III ) ] for 12 hrs . Cell lysates were prepared and subjected to immunoblot analysis with anti - NBR1 and anti - Actin antibodies . Data shown are representative of three separate experiments . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin . Data are shown as means ± s . e . * P < 0 . 05 , and * * P < 0 . 01 as determined by Welch ' 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Immunoblot analysis. Primary hepatocytes were isolated from wild-type (wt), p62f/f; Alb-Cre (p62KO) and Nrf2f/f; Alb-Cre (Nrf2KO) mice. The hepatocytes were challenged with 10 μM sodium arsenite [As (III)] for 12 hrs. Cell lysates were prepared and subjected to immunoblot analysis with anti-NBR1 and anti-Actin antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05, and **P < 0.01 as determined by Welch's t-test.


( B ) Real - time PCR . Total RNAs were prepared from primary hepatocytes described in ( A ) . Values were normalized against the amount of mRNA in each non - treated hepatocytes . The experiments were performed three times . Data are shown as means ± s . e . * * P < 0 . 01 and * * * P < 0 . 001 as determined by Welch ' 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) Real-time PCR. Total RNAs were prepared from primary hepatocytes described in (A). Values were normalized against the amount of mRNA in each non-treated hepatocytes. The experiments were performed three times. Data are shown as means ± s.e. **P < 0.01 and ***P < 0.001 as determined by Welch's t-test.


( C ) Immunofluorescence microscopy . Primary hepatocytes isolated from wild - type mice were challenged with 10 μM sodium arsenite [ As ( III ) ] for 12 hrs and then immunostained with anti - p62 and anti - NBR1 antibodies . Each inset is a magnified image . Bars : 20 µm . The number and size of p62 - bodies were measured in more than 100 cells . Data are shown as means ± s . e . * * * P < 0 . 001 as determined by Welch ' s t - test .

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

(C) Immunofluorescence microscopy. Primary hepatocytes isolated from wild-type mice were challenged with 10 μM sodium arsenite [As (III)] for 12 hrs and then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. ***P < 0.001 as determined by Welch's t-test.


A ) Immunoblot analysis . Primary hepatocytes were isolated from Nbr1f / f mice and then infected with adenovirus GFP or Cre for 48 hrs . Thereafter , the hepatocytes were divided into 3 groups : 1 . Cultured in regular medium ( - ) , 2 . Treated with 10 μM As ( III ) for 12 hr [ As ( III ) ] and 3 . After treated with 10 μM As ( III ) for 12 hr , cultured in regular medium for another 12 hr ( Wash ) . Cell lysates were prepared and subjected to immunoblot analysis with indicated antibodies . Data shown are representative of three separate experiments . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin . Data are shown as means ± s . e . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 as determined by Welch ' 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) Immunoblot analysis. Primary hepatocytes were isolated from Nbr1f/f mice and then infected with adenovirus GFP or Cre for 48 hrs. Thereafter, the hepatocytes were divided into 3 groups: 1. Cultured in regular medium (-), 2. Treated with 10 μM As (III) for 12 hr [As (III)] and 3. After treated with 10 μM As (III) for 12 hr, cultured in regular medium for another 12 hr (Wash). Cell lysates were prepared and subjected to immunoblot analysis with indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05, **P < 0.01, and ***P < 0.001 as determined by Welch's t-test.


B ) Real - time PCR . Total RNAs were prepared from primary hepatocytes described in ( A ) . Values were normalized against the amount of mRNA in the Nbr1f / f hepatocytes expressing GFP under normal culture conditions . The experiments were performed at least three times . Data are shown as means ± s . e . * P < 0 . 05 as determined by Welch ' 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

B) Real-time PCR. Total RNAs were prepared from primary hepatocytes described in (A). Values were normalized against the amount of mRNA in the Nbr1f/f hepatocytes expressing GFP under normal culture conditions. The experiments were performed at least three times. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.


( C ) Immunofluorescence microscopy . Nbr1f / f hepatocytes were infected with LacZ or Cre adenovirus for 48 hrs and , when indicated , challenged with 10 μM As ( III ) for 12 hrs . The cells were then immunostained with anti - p62 and anti - NBR1 antibodies . Each inset is a magnified image . Bars : 20 µm . The number and size of p62 - bodies were measured in more than 100 cells . Data are shown as means ± s . e . * * P < 0 . 01 , and * * * P < 0 . 001 as determined by Welch ' s t - test . ( D ) Role of NBR1 in the p62 - liquid droplets and the p62 - mediated Nrf2 activation . Oxidative stress up - regulates the level of NBR1 , which enhances the phosphorylation and liquid - droplet formation of p62 . The resulting p62 - droplets are resistant to autophagic degradation and serve as signaling nodes for the activation of Nrf2 . Since p62 is one of the targets of Nrf2 , increased level of p62 in cooperation with NBR1 promote the formation of liquid droplets and contributes to the persistent activation of Nrf2 against oxidative stress .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence microscopy. Nbr1f/f hepatocytes were infected with LacZ or Cre adenovirus for 48 hrs and, when indicated, challenged with 10 μM As (III) for 12 hrs. The cells were then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. **P < 0.01, and ***P < 0.001 as determined by Welch's t-test. (D) Role of NBR1 in the p62-liquid droplets and the p62-mediated Nrf2 activation. Oxidative stress up-regulates the level of NBR1, which enhances the phosphorylation and liquid-droplet formation of p62. The resulting p62-droplets are resistant to autophagic degradation and serve as signaling nodes for the activation of Nrf2. Since p62 is one of the targets of Nrf2, increased level of p62 in cooperation with NBR1 promote the formation of liquid droplets and contributes to the persistent activation of Nrf2 against oxidative stress.


( A ) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control of tamoxifen , scale bar 100

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

(A) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control of tamoxifen, scale bar 100 μm


( B Quantification of GLUT1 ( hypoxia marker Control ( n = 5 ) , 2mg ( n = 5 ) , and 5mg ( n = 4 ) . In all cases , bars represent mean ± s . e .

O O O O O O O O O O O O O O O O O O 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 GLUT1 (hypoxia marker Control (n=5), 2mg (n=5), and 5mg (n=4). In all cases, bars represent mean ± s.e.m


( C ) Relative values of protein levels for Glut1 in PDAC tumors assessed by proteomic analysis ( 6 mice per condition and samples were analysed in duplicates )

O O O O O 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 values of protein levels for Glut1 in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates)


, D ) Quantification o CD31 ( endothelial cell marker ) . Control ( n = 5 ) , 2mg ( n = 5 ) , and 5mg ( n = 4 ) . In all cases , bars represent mean ± 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

,D) Quantification o CD31 (endothelial cell marker). Control (n=5), 2mg (n=5), and 5mg (n=4). In all cases, bars represent mean ± s.e.m. (C


( E , F ) Expression levels of DEGs relevant to response to hypoxia ( left ) and blood vessel morphogenesis ( right ) . The values were normalized by tubulin family

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

(E,F) Expression levels of DEGs relevant to response to hypoxia (left) and blood vessel morphogenesis (right). The values were normalized by tubulin family genes


( G ) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control and 5mg of tamoxifen , scale bar 100

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

(G) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control and 5mg of tamoxifen, scale bar 100 μm


( H ) Quantification of HIF - 1A in PDAC tissues Control ( n = 5 ) , and 5mg ( n = 4 ) . In the box - and - whisker plot , the central box represents values from the lower to upper quartile . The middle line represents the mean . The vertical line extends from the minimum to the maximum

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of HIF-1A in PDAC tissues Control (n=5), and 5mg (n=4). In the box-and-whisker plot, the central box represents values from the lower to upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum value


( I ) qPCR levels of HIF - 1A in PSCs , normalized to RPLP0 and relative to

(I) qPCR levels of HIF-1A in PSCs, normalized to RPLP0 and relative to control


( J ) Western blot bands for protein expression in PSCs ( p - Tmod is post - translational modification ) . The plot shows the quantification of the sum of band intensities corresponding to isoform 1 , isoform 2 , isoform 3 , and post - transcriptionally modified HIF - 1A ( n = 8 control and n = 8 tam )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot bands for protein expression in PSCs (p-Tmod is post-translational modification). The plot shows the quantification of the sum of band intensities corresponding to isoform 1, isoform 2, isoform 3, and post-transcriptionally modified HIF-1A (n=8 control and n=8 tam)


( A ) qPCR levels of LOX - L2 in PSCs , normalized to RPLP0 and relative to

(A) qPCR levels of LOX-L2 in PSCs, normalized to RPLP0 and relative to control


( B ) Western blot levels of LOX - L2 in PSCs ( n = 3 experimental replicates , * * 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

(B) Western blot levels of LOX-L2 in PSCs (n=3 experimental replicates, **P<0.01)


( C ) Expression of LOX family genes obtained from RNA - seq data in control and tamoxifen treated PSCs ( n = 3 experimental replicates ) . Expression value was normalized by tubulin family

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

(C) Expression of LOX family genes obtained from RNA-seq data in control and tamoxifen treated PSCs (n = 3 experimental replicates). Expression value was normalized by tubulin family genes


( D ) qPCR levels of LOX family in PSCs , normalized to RPLP0 and relative to

(D) qPCR levels of LOX family in PSCs, normalized to RPLP0 and relative to control


( E ) Relative values of protein levels for LOX members in PDAC tumors assessed by proteomic analysis ( 6 mice per condition and samples were analysed 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

(E) Relative values of protein levels for LOX members in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates


( F ) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle ( control ) , and 2mg and 5mg of tamoxifen , scale bar 50 μm . ( G ) Quantification of LOX - L2 for images in F . n ≥ 4 mice per condition , and n > 10 sections per

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

(F) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle (control), and 2mg and 5mg of tamoxifen, scale bar 50 μm. (G) Quantification of LOX-L2 for images in F. n ≥ 4 mice per condition, and n> 10 sections per animal


( H ) qPCR levels of LOX - L2 and HIF - 1A in PSCs , normalized to RPLP0 and relative to 1

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

(H) qPCR levels of LOX-L2 and HIF-1A in PSCs, normalized to RPLP0 and relative to 1 kPa


( I ) qPCR levels of LOX - L2 and HIF - 1A in PSCs , normalized to RPLP0 and relative to

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

(I) qPCR levels of LOX-L2 and HIF-1A in PSCs, normalized to RPLP0 and relative to control


( J ) Quantification of average forces applied by PSCs on pillars . BBI = blebbistatin . In the box and whisker plot , the central box represents values from the lower and upper quartile . The middle line represents the mean . The vertical line extends from the minimum to the maximum . * * * P < 0 . 001 Three experimental

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

(J) Quantification of average forces applied by PSCs on pillars. BBI = blebbistatin. In the box and whisker plot, the central box represents values from the lower and upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum. ***P<0.001 Three experimental repeats


( K ) qPCR levels of LOX - L2 in PSCs , normalized to RPLP0 ( 60S acidic ribosomal protein P0 ) and relative to

O 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) qPCR levels of LOX-L2 in PSCs, normalized to RPLP0 (60S acidic ribosomal protein P0) and relative to control


( A ) Images of matrigel collagen gels previously remodelled by PSCs , second harmonic generation signal for fibrillar collagen ( green ) and F - actin ( red ) , scale bar 100 μm . ( B ) Fiber thickness colour - code map in a represented through the BoneJ plugin where larger spheres fit along fibers represent greater thickness , scale bar 100 μm . ( C ) SHG fibrillar collagen images used for calculation of alignment through the FFT algorithm . Insets show FFTs of fibrillar collagen - I images , representing alignment with respect to the elliptical distribution of the FFT central maxima . Circular behaviour ( values approaching 1 ) represent no aligned orientation and lower values represent fiber orientation as alignment is displayed as a power distribution orthogonal to the orientation direction . Scale bar 20 μm ( D - F ) Quantification of fiber thickness , length , and alignment for images in A - C . ( G ) Quantification of collagen fiber thickness using the BoneJ algorithm for images in B , H . n ≥ 6 matrices per condition . In the scattered plots each point represents a section . In the box - and - whisker plot , the central box represents values from the lower to upper quartile . The middle line represents the mean . The vertical line extends from the minimum to the maximum value . Histogram bars represent mean ± s . e . m . * * * P < 0 . 001 ( t - test for D , E , F and ; Anova and Tukey " s post hoc test for G ) . Three experimental replicates for all

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Images of matrigel collagen gels previously remodelled by PSCs, second harmonic generation signal for fibrillar collagen (green) and F-actin (red), scale bar 100 μm. (B) Fiber thickness colour-code map in a represented through the BoneJ plugin where larger spheres fit along fibers represent greater thickness, scale bar 100 μm. (C) SHG fibrillar collagen images used for calculation of alignment through the FFT algorithm. Insets show FFTs of fibrillar collagen-I images, representing alignment with respect to the elliptical distribution of the FFT central maxima. Circular behaviour (values approaching 1) represent no aligned orientation and lower values represent fiber orientation as alignment is displayed as a power distribution orthogonal to the orientation direction. Scale bar 20 μm (D-F) Quantification of fiber thickness, length, and alignment for images in A-C. (G) Quantification of collagen fiber thickness using the BoneJ algorithm for images in B,H. n≥6 matrices per condition. In the scattered plots each point represents a section. In the box-and-whisker plot, the central box represents values from the lower to upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum value. Histogram bars represent mean±s.e.m. ***P<0.001 (t-test for D, E, F and ; Anova and Tukey"s post hoc test for G). Three experimental replicates for all p


( A ) qPCR levels of collagen in PSCs , normalized to RPLP0 and relative to

(A) qPCR levels of collagen in PSCs, normalized to RPLP0 and relative to control


( B ) Western blot analysis of collagen normalised to total protein and relative to

(B) Western blot analysis of collagen normalised to total protein and relative to control


( C ) Quantification of time - lapse collagen synthesis and deposition by

(C) Quantification of time-lapse collagen synthesis and deposition by PSCs


( D ) Representative immunofluorescent images used for the quantification in C , scale bar 20 μm , collagen - I was assessed with a specific primary antibody

(D) Representative immunofluorescent images used for the quantification in C, scale bar 20 μm, collagen-I was assessed with a specific primary antibody staining


( E ) Relative values of protein levels for collagen in PDAC tumors assessed by proteomic analysis ( 6 mice per condition and samples were analysed in duplicates )

(E) Relative values of protein levels for collagen in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates)


( F ) qPCR levels of MMP - 2 in PSCs , normalized to RPLP0 and relative to

(F) qPCR levels of MMP-2 in PSCs, normalized to RPLP0 and relative to contro


( G ) MMP - 2 activity on control and tamoxifen treated PSCs assayed by gelatin zymography ; above signal intensity of the representative bands used for the quantification presented in the plot

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(G) MMP-2 activity on control and tamoxifen treated PSCs assayed by gelatin zymography; above signal intensity of the representative bands used for the quantification presented in the plot below


quantification of MMP - 2 levels in PDAC tissues from KPC mice treated with vehicle ( control ) , and 2mg and 5mg of tamoxifen , scale bar 100 μm ( n = 4 animals per condition and n ≥ 5 sections per animal )

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

quantification of MMP-2 levels in PDAC tissues from KPC mice treated with vehicle (control), and 2mg and 5mg of tamoxifen, scale bar 100 μm (n=4 animals per condition and n≥ 5 sections per animal)


Immunohistochemistry image of MMP - 2 levels in PDAC tissues from KPC mice treated with vehicle ( control ) , and 2mg and 5mg of

Immunohistochemistry image of MMP-2 levels in PDAC tissues from KPC mice treated with vehicle (control), and 2mg and 5mg of tamoxife


( J ) Relative values of protein levels for MMP - 2 in PDAC tumors assessed by proteomic analysis ( 6 mice per condition and samples were analysed in duplicates )

O O O O O O O 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) Relative values of protein levels for MMP-2 in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates)


( K ) qPCR levels of MMP - 2 in PSCs , normalized to RPLP0 and relative to 1

(K) qPCR levels of MMP-2 in PSCs, normalized to RPLP0 and relative to 1 kPa


( L ) qPCR levels of MMP - 2 in PSCs , normalized to RPLP0 and relative to control . Histogram bars represent mean ± s . e . m .

(L) qPCR levels of MMP-2 in PSCs, normalized to RPLP0 and relative to control. Histogram bars represent mean±s.e.m.


( A ) qPCR levels of fibronectin ( FN ) , fibronectin extracellular domain A ( FN - EDA ) , and fibronectin extracellular domain B ( FN - EDB ) in PSCs , normalised to RPLP0 and relative to

O O O O O O O O O O O O O O O O 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) qPCR levels of fibronectin (FN), fibronectin extracellular domain A (FN-EDA), and fibronectin extracellular domain B (FN-EDB) in PSCs, normalised to RPLP0 and relative to control


( B ) Western blot analysis of FN normalised to total protein and relative to

(B) Western blot analysis of FN normalised to total protein and relative to control


( C ) Quantification of fibronectin intensity density o FN fluorescence and SHG collagen images for matrigel collagen gels remodelled by

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

(C) Quantification of fibronectin intensity density o FN fluorescence and SHG collagen images for matrigel collagen gels remodelled by PSCs


( D ) FN fluorescence and SHG collagen images for matrigel collagen gels remodelled by

(D) FN fluorescence and SHG collagen images for matrigel collagen gels remodelled by PSCs


( E ) Immunohistochemistry images of PDAC tissues from KPC mice treated with vehicle control , and 2mg , and 5mg of tamoxifen . ( n = 4 animals per condition , and n ≥ 5 sections per animal ) . ( F ) FN fiber thickness colour - code map in E represented through the BoneJ

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

(E) Immunohistochemistry images of PDAC tissues from KPC mice treated with vehicle control, and 2mg, and 5mg of tamoxifen. (n=4 animals per condition, and n ≥ 5 sections per animal). (F) FN fiber thickness colour-code map in E represented through the BoneJ plugin


( G ) Relative values of protein levels for FN in PDAC tumors assessed by proteomic

(G) Relative values of protein levels for FN in PDAC tumors assessed by proteomic analysis


( H - J ) Quantification of fibronectin immunohistochemistry staining , fiber thickness and alignment scored of images presented in e . In all cases , histogram bars represent mean ± s . e . m . ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( t - test for A , B , C ; Anova and Tukey " s post hoc test for H , I , J ) . Scale bar is

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) Quantification of fibronectin immunohistochemistry staining, fiber thickness and alignment scored of images presented in e. In all cases, histogram bars represent mean ± s.e.m.; *P<0.05,**P<0.01,***P<0.001 (t-test for A, B, C; Anova and Tukey"s post hoc test for H, I, J). Scale bar is 1


( A ) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control , and 2mg of tamoxifen , scale bar is 100 μm . Upper panels : Ki67 staining is used as a surrogate of proliferation . White arrows indicate Ki67 positive nuclei in epithelial cells . Lower panels : Cc3 staining shows the cells undergoing caspase - mediated apoptosis . Tamoxifen panels show higher levels of yellow staining , which indicates higher percentage of apoptotic epithelial cells . ( B ) Quantification of staining in panel A ( n = 4 animals per condition , and n ≥ 5 sections per animal , two experimental repetitions ) . Histogram bars represent mean ± s . e . m . ; * * * P < 0 . 001 , t -

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 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

(A) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control, and 2mg of tamoxifen, scale bar is 100 μm. Upper panels: Ki67 staining is used as a surrogate of proliferation. White arrows indicate Ki67 positive nuclei in epithelial cells. Lower panels: Cc3 staining shows the cells undergoing caspase-mediated apoptosis. Tamoxifen panels show higher levels of yellow staining, which indicates higher percentage of apoptotic epithelial cells. (B) Quantification of staining in panel A (n=4 animals per condition, and n≥ 5 sections per animal, two experimental repetitions). Histogram bars represent mean ± s.e.m.; ***P<0.001, t-test


( A , E , F ) Immunofluorescence images of control and tamoxifen treated suit - 2 cells , scale bar is 20 μm . Panel A represents HIF - 1A staining in hypoxia and non - hypoxia conditions , panels E and F show Ki67 and Cc3 staining as markers of proliferation and caspase - mediated apoptosis , respectively . Panel E : Red - F - actin , green - Ki67 , blue - nuclei . Tamoxifen negatively regulates HIF - 1A in hypoxia and non - hypoxia conditions . ( B , C , D ) Quantification of immunofluorescence staining in panels A , E , F . For quantification , eight fields of view ( n > 50 cells ) per condition . Histogram bars represent mean ± s . e . m . ; * * * P < 0 . 001 , t - test . All panels include data collected during 3 independent

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 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

(A, E, F) Immunofluorescence images of control and tamoxifen treated suit-2 cells, scale bar is 20 μm. Panel A represents HIF-1A staining in hypoxia and non-hypoxia conditions, panels E and F show Ki67 and Cc3 staining as markers of proliferation and caspase-mediated apoptosis, respectively. Panel E: Red - F-actin, green - Ki67, blue - nuclei. Tamoxifen negatively regulates HIF-1A in hypoxia and non-hypoxia conditions. (B,C,D) Quantification of immunofluorescence staining in panels A, E, F. For quantification, eight fields of view (n>50 cells) per condition. Histogram bars represent mean ± s.e.m.; ***P<0.001, t-test. All panels include data collected during 3 independent experiments


A Northern blots comparing GlmY and GlmZ levels in wild type strain Z8 and the ∆ rapZ mutant Z28 under normal growth and GlcN6P starvation conditions . Both strains , which also carried a chromosomal glmS ' - lacZ fusion , were treated with 60 μg / ml Nva - FMDP or H2O ( " mock " ) . Samples were harvested hourly for Northern analysis and determination of β - galactosidase activity . Growth curves are shown in Appendix Fig S1 . Blots were re - probed using a 5S rRNA specific probe to provide loading controls .

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

A Northern blots comparing GlmY and GlmZ levels in wild type strain Z8 and the ∆rapZ mutant Z28 under normal growth and GlcN6P starvation conditions. Both strains, which also carried a chromosomal glmS'-lacZ fusion, were treated with 60 μg/ml Nva-FMDP or H2O ("mock"). Samples were harvested hourly for Northern analysis and determination of β-galactosidase activity. Growth curves are shown in Appendix Fig S1. Blots were re-probed using a 5S rRNA specific probe to provide loading controls.


B The purification profile of Strep - RapZ from the ∆ glmS strain Z904 under GlcN6P replete and depletion conditions is shown ( top ) . The cleared lysate ( CL ) , flow through ( FT ) , washing steps ( W ) and the elution fractions ( E1 - 3 ) from StrepTactin affinity chromatography were separated on 12 . 5 % SDS - PAA gels and stained with Coomassie blue . Metabolites were extracted from E2 and analyzed by HILIC - MS / MS . The extracted ion chromatograms of the LC - MS analysis targeting GlcN6P ( retention time 16 . 6 minutes ) are shown below . The samples derived from purification of Strep - RapZ ( panels i and ii ) or Strep - KdpE ( panels iii and iv ) were analyzed with the SRM transition m / z 258 . 1 to m / z 97 in the negative ion mode . The identity of the metabolite detected in panel i was confirmed by adding chemically pure GlcN6P to a final concentration of 100 pg / μl ( panel v ) .

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

B The purification profile of Strep-RapZ from the ∆glmS strain Z904 under GlcN6P replete and depletion conditions is shown (top). The cleared lysate (CL), flow through (FT), washing steps (W) and the elution fractions (E1-3) from StrepTactin affinity chromatography were separated on 12.5 % SDS-PAA gels and stained with Coomassie blue. Metabolites were extracted from E2 and analyzed by HILIC-MS/MS. The extracted ion chromatograms of the LC-MS analysis targeting GlcN6P (retention time 16.6 minutes) are shown below. The samples derived from purification of Strep-RapZ (panels i and ii) or Strep-KdpE (panels iii and iv) were analyzed with the SRM transition m/z 258.1 to m/z 97 in the negative ion mode. The identity of the metabolite detected in panel i was confirmed by adding chemically pure GlcN6P to a final concentration of 100 pg/μl (panel v).


C SPR analysis addressing interaction of RapZ variants with GlcN6P and similar metabolites . The Strep - tagged proteins were captured onto a sensor chip and various concentrations of the respective metabolite ( i . e . 100 , 500 , 1000 , 2500 and 5000 nM ) were injected using a single cycle kinetics approach .

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

C SPR analysis addressing interaction of RapZ variants with GlcN6P and similar metabolites. The Strep-tagged proteins were captured onto a sensor chip and various concentrations of the respective metabolite (i.e. 100, 500, 1000, 2500 and 5000 nM) were injected using a single cycle kinetics approach.


Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions . strains were grown in 96 - well plates and exposed to various degrees of GlcN6P depletion elicited by Nva - FMDP . Cells were harvested at indicated times and the β - galactosidase activities were determined . Strains Z197 and Z360 were used , which harbor glmY ' - lacZ and glmZ ' - lacZ fusions , respectively .

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

Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Strains Z197 and Z360 were used, which harbor glmY'-lacZ and glmZ'-lacZ fusions, respectively.


Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions . strains were grown in 96 - well plates and exposed to various degrees of GlcN6P depletion elicited by Nva - FMDP . Cells were harvested at indicated times and the β - galactosidase activities were determined . Expression of glmY ' - lacZ in strain Z197 and the ∆ rapZ mutant Z225 is compared .

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

Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Expression of glmY'-lacZ in strain Z197 and the ∆rapZ mutant Z225 is compared.


Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions . strains were grown in 96 - well plates and exposed to various degrees of GlcN6P depletion elicited by Nva - FMDP . Cells were harvested at indicated times and the β - galactosidase activities were determined . Strains Z190 and Z201 were addressed , which transcribe glmY ' - lacZ either from the σ54 - promoter or the σ70 promoter , respectively .

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

Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Strains Z190 and Z201 were addressed, which transcribe glmY'-lacZ either from the σ54-promoter or the σ70 promoter, respectively.


Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions . strains were grown in 96 - well plates and exposed to various degrees of GlcN6P depletion elicited by Nva - FMDP . Cells were harvested at indicated times and the β - galactosidase activities were determined . Strains Z190 and the ∆ qseF mutant Z196 are compared , both of which transcribe the glmY ' - lacZ fusion solely from the σ54 promoter .

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

Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Strains Z190 and the ∆qseF mutant Z196 are compared, both of which transcribe the glmY'-lacZ fusion solely from the σ54 promoter.


Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions . strains were grown in 96 - well plates and exposed to various degrees of GlcN6P depletion elicited by Nva - FMDP . Cells were harvested at indicated times and the β - galactosidase activities were determined . Complementation experiment analyzing the requirement of rapZ for glmY expression in cells grown to exponential phase under standard conditions in flask cultures . Strains Z197 and the ∆ rapZ mutant Z225 were used . Tested plasmids were pFDX4291 ( vector control for pFDX4324 and pYG82 = VC1 ) , pFDX4324 ( rapZ ) , pYG82 ( rapZquad ) , pBGG237 ( vector control for pBGG164 = VC2 ) and pBGG164 ( strep - rapZ ) .

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

Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Complementation experiment analyzing the requirement of rapZ for glmY expression in cells grown to exponential phase under standard conditions in flask cultures. Strains Z197 and the ∆rapZ mutant Z225 were used. Tested plasmids were pFDX4291 (vector control for pFDX4324 and pYG82 = VC1), pFDX4324 (rapZ), pYG82 (rapZquad), pBGG237 (vector control for pBGG164 = VC2) and pBGG164 (strep-rapZ).


A BACTH assay addressing interaction of RapZ with QseF and QseE . The following plasmid combinations were tested using reporter strain RH785 ( columns 1 - 6 ) : pBGG352 / pBGG349 , pBGG353 / pBGG348 , pYG199 / pBGG349 , pYG246 / pBGG348 , pKT25 / pUT18C ( negative control ) , pKT25 - zip / pUT18C - zip ( positive control ) . , β - galactosidase activities are presented as mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 BACTH assay addressing interaction of RapZ with QseF and QseE. The following plasmid combinations were tested using reporter strain RH785 (columns 1-6): pBGG352/pBGG349, pBGG353/pBGG348, pYG199/pBGG349, pYG246/pBGG348, pKT25/pUT18C (negative control), pKT25-zip/pUT18C-zip (positive control). , β-galactosidase activities are presented as mean ± SD.


B SPR spectroscopy experiments addressing interaction of RapZ with the cytoplasmic part ( aa 196 - 475 ) of kinase QseE and response regulator QseF . Strep - RapZ was captured onto a sensor chip and increasing concentrations of QseE ' - His10 or QseF - His10 were injected .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 SPR spectroscopy experiments addressing interaction of RapZ with the cytoplasmic part (aa 196-475) of kinase QseE and response regulator QseF. Strep-RapZ was captured onto a sensor chip and increasing concentrations of QseE'-His10 or QseF-His10 were injected.


C To assess the role of RapZ for QseF activity , β - galactosidase activities were determined from strains Z196 ( ∆ qseF ) and Z1110 ( ∆ qseF ∆ rapZ ) at indicated times during growth . Strains harbored the glmY ' - lacZ fusion that is solely expressed from the σ54 promoter and the following plasmids : pKESK23 ( VC = vector control ; black and grey ; note that activities are too low for display ) , pYG89 ( qseF , green ) , pYG90 ( qseF - D56E , purple ) , pYG93 ( qseF - D56A , red ) . , β - galactosidase activities are presented as mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 To assess the role of RapZ for QseF activity, β-galactosidase activities were determined from strains Z196 (∆qseF) and Z1110 (∆qseF ∆rapZ) at indicated times during growth. Strains harbored the glmY'-lacZ fusion that is solely expressed from the σ54 promoter and the following plasmids: pKESK23 (VC = vector control; black and grey; note that activities are too low for display), pYG89 (qseF, green), pYG90 (qseF-D56E, purple), pYG93 (qseF-D56A, red). , β-galactosidase activities are presented as mean ± SD.


D In vitro phosphorylation assays addressing autophosphorylation of 1 μM His10 - tagged QseE ' ( aa 196 - 475 ) in presence of various concentrations of RapZ . Samples were removed following [ γ - 32P ] - ATP addition at indicated times and separated on 12 . 5 % SDS - PAA gels , which were analyzed by phospho imaging .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 In vitro phosphorylation assays addressing autophosphorylation of 1 μM His10-tagged QseE' (aa 196-475) in presence of various concentrations of RapZ. Samples were removed following [γ-32P]-ATP addition at indicated times and separated on 12.5 % SDS-PAA gels, which were analyzed by phospho imaging.


E Analysis of QseE ' autophosphorylation ( lanes 1 - 6 ) and phosphoryl group transfer to QseF ( lanes 7 - 12 ) in absence or presence of 5 μM Strep - RapZ . To assess phosphoryl group transfer , 1 μM QseF - His10 was added to the assay .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Analysis of QseE' autophosphorylation (lanes 1-6) and phosphoryl group transfer to QseF (lanes 7-12) in absence or presence of 5 μM Strep-RapZ. To assess phosphoryl group transfer, 1 μM QseF-His10 was added to the assay.


F To analyse the role of GlcN6P , 5 μM RapZ or the equivalent volume of buffer was pre - incubated with the indicated GlcN6P concentration for 5 min and subsequently 1 μM QseE ' - His10 was added . Following an additional incubation for 5 min , [ γ - 32P ] - ATP was added and the reactions were stopped after 1 min .

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F To analyse the role of GlcN6P, 5 μM RapZ or the equivalent volume of buffer was pre-incubated with the indicated GlcN6P concentration for 5 min and subsequently 1 μM QseE'-His10 was added. Following an additional incubation for 5 min, [γ-32P]-ATP was added and the reactions were stopped after 1 min.


A β - Galactosidase activities of strains Z197 ( wild type ) and Z1118 ( ∆ glmY ∆ glmZ ) , which carry the chromosomal glmY ' - lacZ fusion , were determined during growth .

O O O O O O O O O O O O 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 β-Galactosidase activities of strains Z197 (wild type) and Z1118 (∆glmY ∆glmZ), which carry the chromosomal glmY'-lacZ fusion, were determined during growth.


B Strains Z197 ( wild type ) and Z225 ( ∆ rapZ ) were transformed with the following plasmids expressing the mentioned sRNAs : pBR - plac ( vector control = VC ) , pYG83 ( glmY ) , pYG84 ( glmZ ) and pSD69 ( gcvB ) . sRNA expression was induced with 1 mM IPTG and β - galactosidase activities were determined in the exponential growth phase .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Strains Z197 (wild type) and Z225 (∆rapZ) were transformed with the following plasmids expressing the mentioned sRNAs: pBR-plac (vector control = VC), pYG83 (glmY), pYG84 (glmZ) and pSD69 (gcvB). sRNA expression was induced with 1 mM IPTG and β-galactosidase activities were determined in the exponential growth phase.


C To assess the impact of GlmY * and GlmZ on stimulation of QseE ' autophosphorylation by RapZ , 1 μM QseE ' - His10 was incubated with [ γ - 32P ] - ATP in absence or presence of 5 μM Strep - RapZ and / or the sRNAs GlmY * , GlmZ and GcvB . In lanes 3 - 11 QseE ' was co - incubated with RapZ as well as the indicated sRNAs provided at 0 . 5 μM , 1 . 25 μM and 2 . 5 μM . In lanes 14 - 16 , QseE ' was incubated with 2 . 5 μM of each sRNA without RapZ . Samples were removed 1 min after addition of [ γ - 32P ] - ATP and separated on 12 . 5 % SDS - PAA gels , which were analyzed by phospho imaging .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 To assess the impact of GlmY* and GlmZ on stimulation of QseE' autophosphorylation by RapZ, 1 μM QseE'-His10 was incubated with [γ-32P]-ATP in absence or presence of 5 μM Strep-RapZ and/or the sRNAs GlmY*, GlmZ and GcvB. In lanes 3-11 QseE' was co-incubated with RapZ as well as the indicated sRNAs provided at 0.5 μM, 1.25 μM and 2.5 μM. In lanes 14-16, QseE' was incubated with 2.5 μM of each sRNA without RapZ. Samples were removed 1 min after addition of [γ-32P]-ATP and separated on 12.5 % SDS-PAA gels, which were analyzed by phospho imaging.


Northern blot experiments assessing the half - lives of GlmY * and GlmZ under normal growth and GlcN6P starvation conditions . Bacterial cultures were either treated with 100 μg / ml Nva - FMDP or H2O ( mock ) . Transcription was stopped by rifampicin addition when cultures attained OD600 = 1 . 0 and samples were removed at indicated times for Northern analysis . A Analysis of GlmY * and GlmZ decay in the wild type strain Z8 . B Analysis of GlmY * and GlmZ decay in the ∆ rapZ mutant Z28 . C Semi - logarithmic plots of GlmY * and full - length GlmZ decay for half - life determination .

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

Northern blot experiments assessing the half-lives of GlmY* and GlmZ under normal growth and GlcN6P starvation conditions. Bacterial cultures were either treated with 100 μg/ml Nva-FMDP or H2O (mock). Transcription was stopped by rifampicin addition when cultures attained OD600 = 1.0 and samples were removed at indicated times for Northern analysis. A Analysis of GlmY* and GlmZ decay in the wild type strain Z8. B Analysis of GlmY* and GlmZ decay in the ∆rapZ mutant Z28. C Semi-logarithmic plots of GlmY* and full-length GlmZ decay for half-life determination.


Northern blot experiments ( A - C ) assessing GlmY * half - life under GlcN6P replete ( + GlcN ) and depletion conditions ( - GlcN ) . Transcription was stopped by addition of rifampicin and samples were harvested at indicated times for Northern analysis . A Analysis of the ∆ glmS strain Z1126 . The GlcN6P depleted culture was split 8 min after rifampicin addition and one of the sub - cultures was resupplied with GlcN ( indicated by arrow ) . B The ∆ glmS ∆ rapZ double mutant Z1127 was tested . C Semi - logarithmic plots of GlmY * decay for half - life determination .

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

Northern blot experiments (A-C) assessing GlmY* half-life under GlcN6P replete (+GlcN) and depletion conditions (-GlcN). Transcription was stopped by addition of rifampicin and samples were harvested at indicated times for Northern analysis. A Analysis of the ∆glmS strain Z1126. The GlcN6P depleted culture was split 8 min after rifampicin addition and one of the sub-cultures was resupplied with GlcN (indicated by arrow). B The ∆glmS ∆rapZ double mutant Z1127 was tested. C Semi-logarithmic plots of GlmY* decay for half-life determination.


D EMSA experiments addressing the role of GlcN6P for GlmY * / RapZ interaction . Radiolabelled GlmY * was incubated with incremental concentrations of RapZ ( left panel ) or RapZ - CTD ( right panel ) in absence or presence of 7 . 5 mM GlcN6P . Binding reactions were separated on native PAA gels and analyzed by phospho - imaging . The RapZ / GlmY * complex is indicated by an arrow .

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 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

D EMSA experiments addressing the role of GlcN6P for GlmY*/RapZ interaction. Radiolabelled GlmY* was incubated with incremental concentrations of RapZ (left panel) or RapZ-CTD (right panel) in absence or presence of 7.5 mM GlcN6P. Binding reactions were separated on native PAA gels and analyzed by phospho-imaging. The RapZ/GlmY* complex is indicated by an arrow.


E EMSA following incubation of GlmY * with 1200 nM RapZ in presence of various GlcN6P concentrations ranging from 0 ( lane 2 ) to 8 mM ( lane 10 ) . The fraction of GlmY * remaining in the gel pocket is marked with an asterisk . The RapZ / GlmY * complex is indicated by an arrow .

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

E EMSA following incubation of GlmY* with 1200 nM RapZ in presence of various GlcN6P concentrations ranging from 0 (lane 2) to 8 mM (lane 10). The fraction of GlmY* remaining in the gel pocket is marked with an asterisk. The RapZ/GlmY* complex is indicated by an arrow.


F EMSA following incubation of GlmY * with 1200 nM RapZ in absence or presence of 7 . 5 mM of the indicated metabolite . The RapZ / GlmY * complex is indicated by an arrow .

O O O O O O O O O O O O O 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 EMSA following incubation of GlmY* with 1200 nM RapZ in absence or presence of 7.5 mM of the indicated metabolite. The RapZ/GlmY* complex is indicated by an arrow.


B ChIP gel images showing the relative enrichment by H3K4me3 in controls ( n = 9 ) and PD patients ( n = 18 ) . PCR amplified a 188 - bp region of SNCA from intron 1 where H3K4me3 peak was at its optimum . Mouse IgG ( mIgG ) was used as control and the bands were normalized by unbiased amplification from respective inputs . Data information : Data represent mean ± standard error of the mean .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ChIP gel images showing the relative enrichment by H3K4me3 in controls (n=9) and PD patients (n= 18). PCR amplified a 188-bp region of SNCA from intron 1 where H3K4me3 peak was at its optimum. Mouse IgG (mIgG) was used as control and the bands were normalized by unbiased amplification from respective inputs. Data information: Data represent mean ± standard error of the mean.


C The normalized levels of α - synuclein in the entire cohort were divided into high ( n = 12 ) and low levels . The cut off value for determining the threshold was set at the median from all the subjects . The high levels of α - synuclein were plotted with corresponding H3K4me3 values of those subjects . The corresponding ID of each sample is shown next to each point in the graph . There was a significant correlation between high levels of α - synuclein and H3K4me3 . Data information : Data represent mean ± standard error of the mean . Spearman rank correlation test was performed in ( C ) . Two tailed p value was calculated with Fisher ' s exact p .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The normalized levels of α-synuclein in the entire cohort were divided into high (n = 12) and low levels. The cut off value for determining the threshold was set at the median from all the subjects. The high levels of α-synuclein were plotted with corresponding H3K4me3 values of those subjects. The corresponding ID of each sample is shown next to each point in the graph. There was a significant correlation between high levels of α-synuclein and H3K4me3. Data information: Data represent mean ± standard error of the mean. Spearman rank correlation test was performed in (C). Two tailed p value was calculated with Fisher's exact p.


D The graph represents the statistically significant difference of relative H3K4me3 enrichment between PD ( n = 7 ) and controls ( n = 6 ) . Neuronal nuclei from PD brain samples show significantly higher enrichment of H3K4me3 at SNCA intron 1 ( p = 0 . 01 ) compared to controls . * P < 0 . 05 . Data were analyzed using non - parametric t - test followed by Mann - Whitney post - hoc corrections . Two - tailed p - values were calculated for all . Data information : Data represent mean ± standard error of the mean .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The graph represents the statistically significant difference of relative H3K4me3 enrichment between PD (n=7) and controls (n=6). Neuronal nuclei from PD brain samples show significantly higher enrichment of H3K4me3 at SNCA intron 1 (p=0.01) compared to controls. *P < 0.05. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. Two-tailed p-values were calculated for all. Data information: Data represent mean ± standard error of the mean.


The corresponding α - synuclein levels C ) were evaluated in SH - SY5Y cells expressing dCas9 - 5xGCN4 with ( sgA - dCas9 ) or without scFV - sfGFP - JARID1A ( sgA - dCas9 - JA ) . C α - SYN levels in SH - SY5Y cells were also evaluated using western blot . The levels of α - SYN was normalized to β - actin . Significant reduction of α - SYN was observed in cells expressing JARID1A . Three independent repeats were performed . Percentage of reduction is shown in the graph . * * P < 0 . 01 . Data were analyzed using non - parametric t - test followed by Mann - Whitney post - hoc corrections . Two - tailed p - values were calculated . Data information : All data are presented as mean ± SEM .

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

The corresponding α-synuclein levels C) were evaluated in SH-SY5Y cells expressing dCas9-5xGCN4 with (sgA-dCas9) or without scFV-sfGFP-JARID1A (sgA-dCas9-JA). C α-SYN levels in SH-SY5Y cells were also evaluated using western blot. The levels of α-SYN was normalized to β-actin. Significant reduction of α-SYN was observed in cells expressing JARID1A. Three independent repeats were performed. Percentage of reduction is shown in the graph. **P <0.01. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. Two-tailed p-values were calculated. Data information: All data are presented as mean ± SEM.


E ChIP on H3K4me3 at the SNCA promoter between sPD1 - 1 lines after locus - specific epigenomic modulation . Differentiated cells were transiently transfected with either sgA - dCas9 5xGCN4 - scFV - JARID1A or sg A - dCas9 5xGCN4 - scFV - empty backbone vectors . A significant reduction in H3K4me3 at the SNCA promoter was observed in cells transfected with JARID1A . Data information : Three independent repeats were performed for both ChIP experiments . * P < 0 . 05 ; * * * * P < 0 . 0001 . Data were analyzed using non - parametric t - test followed by Mann - Whitney post - hoc corrections . One - tailed p - values were calculated for ChIP

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ChIP on H3K4me3 at the SNCA promoter between sPD1-1 lines after locus-specific epigenomic modulation. Differentiated cells were transiently transfected with either sgA-dCas9 5xGCN4-scFV-JARID1A or sg A-dCas9 5xGCN4-scFV-empty backbone vectors. A significant reduction in H3K4me3 at the SNCA promoter was observed in cells transfected with JARID1A. Data information: Three independent repeats were performed for both ChIP experiments. *P < 0.05; ****P<0.0001. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. One-tailed p-values were calculated for ChIP analysis


F Western blot analysis of α - synuclein ( α - SYN ) levels in the cells under the same conditions as analyzed in ( E ) . A significant decrease ( 56 - 66 % ) in α - SYN levels was observed in cells transfected with JARID1A . The normalized and relative expression of α - SYN in JARID1A transfected cells are shown as a percentage of control ( transfected by empty backbone vector ) . Data information : Three independent repeats were performed for western blot experiments . * P < 0 . 05 ; * * * * P < 0 . 0001 . Data were analyzed using non - parametric t - test followed by Mann - Whitney post - hoc corrections . two - tailed p - value was calculated for western blot experiments . All data are presented as mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot analysis of α-synuclein (α-SYN) levels in the cells under the same conditions as analyzed in (E). A significant decrease (56-66%) in α-SYN levels was observed in cells transfected with JARID1A. The normalized and relative expression of α-SYN in JARID1A transfected cells are shown as a percentage of control (transfected by empty backbone vector). Data information: Three independent repeats were performed for western blot experiments. *P < 0.05; ****P<0.0001. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. two-tailed p-value was calculated for western blot experiments. All data are presented as mean ± SEM.


B - Congo red plates with colonies of E . coli strain MC4100 relA + and its target mutation csgD : M4 - ompR : mut2 derivative , harboring sRNA - producing plasmids pOmrA , pOmrB , or an empty vector ( pControl ) . Incubation at 28 ° C for 48 h .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - Congo red plates with colonies of E. coli strain MC4100 relA+ and its target mutation csgD:M4-ompR:mut2 derivative, harboring sRNA-producing plasmids pOmrA, pOmrB, or an empty vector (pControl). Incubation at 28°C for 48 h.


B - Relative fluorescence / OD600 of cells of E . coli strain MC4100 relA + ∆ omrAB or MC4100 relA + ∆ omrAB ∆ hfq harboring the translational fusion plasmid pDgcM : : GFP in combination with plasmids expressing OmrA , OmrB , or an empty control vector , after 16 h of growth at 37 ° C . Error bars SD , N = 3 . Average fluorescence intensities ( a . u . ) / OD600 used to calculate ratios Note that absolute dgcM : : gfp expression values are ≈ 3 - 4 - fold lower in the ∆ hfq

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 fluorescence/OD600 of cells of E. coli strain MC4100 relA+ ∆omrAB or MC4100 relA+ ∆omrAB ∆hfq harboring the translational fusion plasmid pDgcM::GFP in combination with plasmids expressing OmrA, OmrB, or an empty control vector, after 16 h of growth at 37°C. Error bars SD, N=3. Average fluorescence intensities (a.u.)/ OD600 used to calculate ratios Note that absolute dgcM::gfp expression values are ≈3-4-fold lower in the ∆hfq strain


C - Western blot analysis of cell lysates from strains MC4100 relA + ∆ omrAB harboring plasmids pOmrA , pOmrB , or mutant variants as indicated , in combination with a plasmid expressing C - terminal FLAG - tagged dgcM or dgcM _ M2 . As a loading control , part of a protein - stained gel is shown below .

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

C - Western blot analysis of cell lysates from strains MC4100 relA+ ∆omrAB harboring plasmids pOmrA, pOmrB, or mutant variants as indicated, in combination with a plasmid expressing C-terminal FLAG-tagged dgcM or dgcM_M2. As a loading control, part of a protein-stained gel is shown below.


A - Toeprinting showing inhibition of initiation complex formation on a truncated dgcM mRNA ( 210 nt from + 1 ) by OmrA and OmrB . Inclusion of 30S ribosomes , tRNAfmet , OmrA , and OmrB is indicated . The unrelated IstR - 1 sRNA is a negative control . The 30S / tRNAfmet - dependent reverse transcription block ( toeprint ) , is indicated by an arrow . Sequencing reactions were loaded next to the primer extension products ( UCGA ) . The AUG start codon is indicated by asterisks .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - Toeprinting showing inhibition of initiation complex formation on a truncated dgcM mRNA (210 nt from +1) by OmrA and OmrB. Inclusion of 30S ribosomes, tRNAfmet, OmrA, and OmrB is indicated. The unrelated IstR-1 sRNA is a negative control. The 30S/ tRNAfmet-dependent reverse transcription block (toeprint), is indicated by an arrow. Sequencing reactions were loaded next to the primer extension products (UCGA). The AUG start codon is indicated by asterisks.


B - In vitro translation of dgcM : : 3xflag mRNA in the presence or absence of OmrA / OmrB , with or without purified Hfq protein , was carried out As an internal control , the non - target ompA : : 3xflag mRNA was included . The Western blot shows DgcM - 3xFLAG and OmpA - 3xFLAG protein levels .

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

B - In vitro translation of dgcM::3xflag mRNA in the presence or absence of OmrA/ OmrB, with or without purified Hfq protein, was carried out As an internal control, the non-target ompA::3xflag mRNA was included. The Western blot shows DgcM-3xFLAG and OmpA-3xFLAG protein levels.


A - 5 ' end - labeled dgcM mRNA ( 10 nM ) was subjected to probing All components added to the labeled RNA are indicated above the autoradiogram . When added , Hfq hexamers were at 50 nM , the sRNAs OmrA and OmrB at 250 nM . The accessibility of each nucleotide was probed with water ( lanes 1 - 4 , negative control for RNA integrity ) , RNase T1 , or lead ( II ) acetate . Lane T1 : RNase T1 under denaturing conditions . Lane OH : alkaline ladders . Binding sites of Hfq ( green arrows and boxes ) , and site 1 ( red arrows and boxes ) and 2 ( blue arrows and boxes ) of the sRNAs , are highlighted . Note that site 2 is bound differently by OmrA and OmrB ( blue boxes on gel ) . Black boxes indicate Hfq - dependent T1 cleavage enhancements .

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

A - 5' end-labeled dgcM mRNA (10 nM) was subjected to probing All components added to the labeled RNA are indicated above the autoradiogram. When added, Hfq hexamers were at 50 nM, the sRNAs OmrA and OmrB at 250 nM. The accessibility of each nucleotide was probed with water (lanes 1-4, negative control for RNA integrity), RNase T1, or lead(II) acetate. Lane T1: RNase T1 under denaturing conditions. Lane OH: alkaline ladders. Binding sites of Hfq (green arrows and boxes), and site 1 (red arrows and boxes) and 2 (blue arrows and boxes) of the sRNAs, are highlighted. Note that site 2 is bound differently by OmrA and OmrB (blue boxes on gel). Black boxes indicate Hfq-dependent T1 cleavage enhancements.


Mobility shift assay with end - labeled dgcM RNA ( 210 nt species from + 1 ) in the presence or absence of unlabeled OmrA or OmrB . When indicated , single - stranded DNA oligos were pre - annealed to site 1 ( dgcM _ site1 ) , site 2 ( dgcM _ site2 ) , or both , before addition of sRNAs . The additions for each sample are indicated above the Figure , and the identities of the nucleic acid complexes are indicated on the left for guidance .

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

Mobility shift assay with end-labeled dgcM RNA (210 nt species from +1) in the presence or absence of unlabeled OmrA or OmrB. When indicated, single-stranded DNA oligos were pre-annealed to site 1 (dgcM_site1), site 2 (dgcM_site2), or both, before addition of sRNAs. The additions for each sample are indicated above the Figure, and the identities of the nucleic acid complexes are indicated on the left for guidance.


Western blots showing DgcM - 3xFLAG translated in vitro in the presence or absence of OmrA or OmrB . A - The inclusion of purified wild - type or mutant Hfq proteins is indicated , and the translation assay was conducted The different Hfq interaction site mutant proteins are indicated . Data information : Quantification of DgcM translation rates , relative to that of the unaffected OmpA control , is shown below the gels . The ratio of DgcM / OmpA signal was set to unity for the lane lacking sRNA in each set of three combinations ( - sRNA , + OmrA , + OmrB ) , indicated by " 1 . 00 * " . Effects due to OmrA / OmrB addition can be seen by comparing lanes 2 and 3 to lane 1 in each set .

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

Western blots showing DgcM-3xFLAG translated in vitro in the presence or absence of OmrA or OmrB. A - The inclusion of purified wild-type or mutant Hfq proteins is indicated, and the translation assay was conducted The different Hfq interaction site mutant proteins are indicated. Data information: Quantification of DgcM translation rates, relative to that of the unaffected OmpA control, is shown below the gels. The ratio of DgcM/ OmpA signal was set to unity for the lane lacking sRNA in each set of three combinations (-sRNA, +OmrA, +OmrB), indicated by "1.00*". Effects due to OmrA/ OmrB addition can be seen by comparing lanes 2 and 3 to lane 1 in each set.


Western blots showing DgcM - 3xFLAG translated in vitro in the presence or absence of OmrA or OmrB . B - An ssDNA - oligo ( Hfq _ BS that base - pairs specifically to the Hfq binding site on dgcM mRNA ( nt 42 - 66 ) was preannealed to the mRNA prior to OmrA / B addition and translation . OmpA - 3xFLAG was included as internal control . Data information : Quantification of DgcM translation rates , relative to that of the unaffected OmpA control , is shown below the gels . The ratio of DgcM / OmpA signal was set to unity for the lane lacking sRNA in each set of three combinations ( - sRNA , + OmrA , + OmrB ) , indicated by " 1 . 00 * " . Effects due to OmrA / OmrB addition can be seen by comparing lanes 2 and 3 to lane 1 in each set .

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

Western blots showing DgcM-3xFLAG translated in vitro in the presence or absence of OmrA or OmrB. B - An ssDNA-oligo (Hfq_BS that base-pairs specifically to the Hfq binding site on dgcM mRNA (nt 42-66) was preannealed to the mRNA prior to OmrA/B addition and translation. OmpA-3xFLAG was included as internal control. Data information: Quantification of DgcM translation rates, relative to that of the unaffected OmpA control, is shown below the gels. The ratio of DgcM/ OmpA signal was set to unity for the lane lacking sRNA in each set of three combinations (-sRNA, +OmrA, +OmrB), indicated by "1.00*". Effects due to OmrA/ OmrB addition can be seen by comparing lanes 2 and 3 to lane 1 in each set.


A - Histograms show flow cytometry analysis of exponentially growing cells of E . coli strain MC4100 relA - , and its mutant derivatives hfq - F42A ( hfq - prox ) , hfq - Y25D ( hfq - dist ) , hfq - R16S _ R17A _ R19A _ K47E ( hfq - rim ) , ∆ hfq , harboring the translational fusion plasmid pDgcM : : GFP in combination with pOmrA , pOmrB , or a control vector . Data was obtained on a MACSQuant VYB instrument .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - Histograms show flow cytometry analysis of exponentially growing cells of E. coli strain MC4100 relA-, and its mutant derivatives hfq-F42A (hfq-prox), hfq-Y25D (hfq-dist), hfq-R16S_R17A_R19A_K47E (hfq-rim), ∆hfq, harboring the translational fusion plasmid pDgcM::GFP in combination with pOmrA, pOmrB, or a control vector. Data was obtained on a MACSQuant VYB instrument.


B - Northern blot analysis of total RNA isolated from the same cultures at the same time point as used for flow cytometry , probed for OmrA ( strains containing pOmrA ) or OmrB ( strains containing pOmrB ) . 5S rRNA was used as loading control .

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B - Northern blot analysis of total RNA isolated from the same cultures at the same time point as used for flow cytometry, probed for OmrA (strains containing pOmrA) or OmrB (strains containing pOmrB). 5S rRNA was used as loading control.


The upper panel shows strength of association of Affymetrix 500K SNPs in the region , with combined P values shown in red for replicated SNPs . The lower panel is a recombination map showing how the signals are clearly demarcated by recombination hotspots . Nearby genes are shown in yellow .

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The upper panel shows strength of association of Affymetrix 500K SNPs in the region, with combined P values shown in red for replicated SNPs. The lower panel is a recombination map showing how the signals are clearly demarcated by recombination hotspots. Nearby genes are shown in yellow.


Representative images of EGFP - Nix - FKBP ( green ) Rapalog - induced recruitment to mtKeima labeled mitochondria ( Keima excitation 488 , cyan ) and subsequent mitophagy ( Keima excitation 561 , magenta ) . Scale bar : 10 µm . Data Information : Area shading indicates standard deviation between wells . Over one thousand cells quantified per condition .

Representative images of EGFP-Nix-FKBP (green) Rapalog-induced recruitment to mtKeima labeled mitochondria (Keima excitation 488, cyan) and subsequent mitophagy (Keima excitation 561, magenta). Scale bar: 10 µm. Data Information: Area shading indicates standard deviation between wells. Over one thousand cells quantified per condition.


Confirmation of decreased mitochondrial proteins in WT but not in FIP200 ko cells expressing CID Nix either untreated or treated for 24 hours with Rapalog by immunoblotting . Quantification of MTCO2 relative to Actin over three independent experiments after treatment with Rapalog over 24 hours by immunoblotting . Cells in this figure express mtKeima - P2A - FRB - Fis1tTand EGFP - Nix1 - 188 - FKBP . Data Information : Area shading indicates standard deviation between wells . Over one thousand cells quantified per condition . Statistics performed by two - way ANOVA ; * * indicates a p - value < 0 . 01 , * * * indicates a p - value < 0 . 001 . Experiments repeated at least 3 times .

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Confirmation of decreased mitochondrial proteins in WT but not in FIP200 ko cells expressing CID Nix either untreated or treated for 24 hours with Rapalog by immunoblotting. Quantification of MTCO2 relative to Actin over three independent experiments after treatment with Rapalog over 24 hours by immunoblotting. Cells in this figure express mtKeima-P2A-FRB-Fis1tTand EGFP-Nix1-188-FKBP. Data Information: Area shading indicates standard deviation between wells. Over one thousand cells quantified per condition. Statistics performed by two-way ANOVA; ** indicates a p-value<0.01, *** indicates a p-value<0.001. Experiments repeated at least 3 times.


Mitophagy time course by mtKeima of different Nix truncation mutants recruited with CID , measured by percentage of Keima positive pixels above threshold . Data Information : Area shading indicates standard deviation between wells . Over one thousand cells quantified per condition . Experiments repeated at least 3 times .

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Mitophagy time course by mtKeima of different Nix truncation mutants recruited with CID, measured by percentage of Keima positive pixels above threshold. Data Information: Area shading indicates standard deviation between wells. Over one thousand cells quantified per condition. Experiments repeated at least 3 times.


Mitophagy by mtKeima of Nix1 - 70 when treated with Torin , Rapalog , or both compared to control . Mitophagy by mtKeima of Nix1 - 87 when treated with Torin , Rapalog , or both compared to control . Data Information : Area shading indicates standard deviation between wells . Over one thousand cells quantified per condition . Experiments repeated at least 3 times .

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Mitophagy by mtKeima of Nix1-70 when treated with Torin, Rapalog, or both compared to control. Mitophagy by mtKeima of Nix1-87 when treated with Torin, Rapalog, or both compared to control. Data Information: Area shading indicates standard deviation between wells. Over one thousand cells quantified per condition. Experiments repeated at least 3 times.


Volcano plot generated from one - tailed t - test of proteins that interact with Nix1 - 87 more so than Nix1 - 70 by IP - mass spectrometry . Red dots indicate proteins over 2 - fold enrichment and p - value < 0 . 05 Data Information : Statistics : one - tailed unpaired Student ' s t - test . Fold change calculated from spectral counts ( sc ) by ( sc1 - 87 + 1 ) / ( sc1 - 70 + 1 ) so that zero values could be included .

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Volcano plot generated from one-tailed t-test of proteins that interact with Nix1-87 more so than Nix1-70 by IP-mass spectrometry. Red dots indicate proteins over 2-fold enrichment and p-value<0.05 Data Information: Statistics: one-tailed unpaired Student's t-test. Fold change calculated from spectral counts (sc) by (sc1-87+1)/(sc1-70+1) so that zero values could be included.


IP western blot of FKBP - EGFP - Nix1 - 188 , Nix1 - 87 , or Nix1 - 70 , comparing 1 % input to GFP - pulldown and blotted against GFP or WIPI2 after 4 hours of Rapalog . Data Information : Statistics : one - tailed unpaired Student ' s t - test . Fold change calculated from spectral counts ( sc ) by ( sc1 - 87 + 1 ) / ( sc1 - 70 + 1 ) so that zero values could be included . IP Experiments repeated at least 3 times .

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IP western blot of FKBP-EGFP-Nix1-188, Nix1-87, or Nix1-70, comparing 1% input to GFP-pulldown and blotted against GFP or WIPI2 after 4 hours of Rapalog. Data Information: Statistics: one-tailed unpaired Student's t-test. Fold change calculated from spectral counts (sc) by (sc1-87+1)/(sc1-70+1) so that zero values could be included. IP Experiments repeated at least 3 times.


HeLa cells expressing FKBP - EGFP - p62327 - 348 or FKBP - EGFP - WIPI2b were treated for 16 hours with phenanthroline to induce endogenous Nix and BNIP3 expression , then underwent a GFP - pulldown and compared to 1 % input by immunoblot . Data Information IP Experiments repeated at least 3 times .

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HeLa cells expressing FKBP-EGFP- p62327-348 or FKBP-EGFP-WIPI2b were treated for 16 hours with phenanthroline to induce endogenous Nix and BNIP3 expression, then underwent a GFP-pulldown and compared to 1% input by immunoblot. Data Information IP Experiments repeated at least 3 times.


IP western blot of FKBP - EGFP - Nix1 - 188 compared to Nix60 - 188 , comparing 1 % input to GFP - pulldown and blotted against GFP or WIPI2 after 4 hours of Rapalog . Data Information IP Experiments repeated at least 3 times .

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IP western blot of FKBP-EGFP-Nix1-188 compared to Nix60-188, comparing 1% input to GFP-pulldown and blotted against GFP or WIPI2 after 4 hours of Rapalog. Data Information IP Experiments repeated at least 3 times.


Representative images of WT or 6ko HeLa cells expressing EGFP - Nix1 - 188 - FKBP ( green ) , mtKeima , and FRB - Fis1T with or without 4 hours of Rapalog , immunostained for endogenous Tom20 ( red ) and WIPI2 ( white ) . Scale bar : 50µm . Automated puncta counting of WIPI2 in WT or 6ko cells normalized to cell number in control conditions or after 4 hours with Rapalog . Data Information : Error bars indicate standard deviation . Statistics performed by two - way ANOVA ; * * indicates a p - value < 0 . 01 , * * * indicates a p - value < 0 . 001 . ns indicates not significant . Over one thousand cells quantified per condition . Experiments repeated at least 3 times .

O O O O O O O O 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 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

Representative images of WT or 6ko HeLa cells expressing EGFP-Nix1-188-FKBP (green), mtKeima, and FRB-Fis1T with or without 4 hours of Rapalog, immunostained for endogenous Tom20 (red) and WIPI2 (white). Scale bar: 50µm. Automated puncta counting of WIPI2 in WT or 6ko cells normalized to cell number in control conditions or after 4 hours with Rapalog. Data Information: Error bars indicate standard deviation. Statistics performed by two-way ANOVA; ** indicates a p-value<0.01, *** indicates a p-value<0.001. ns indicates not significant. Over one thousand cells quantified per condition. Experiments repeated at least 3 times.


Representative images of WT or WIPI2 ko HeLa cells expressing EGFP - Nix1 - 188 - FKBP ( green ) , mtKeima , and FRB - Fis1T with or without 4 hours of Rapalog immunostained for endogenous Tom20 ( red ) and GABARAP ( white ) . Scale bar : 50µm . Automated puncta counting of GABARAP in WT or WIPI2 ko cells normalized to cell number in control conditions or after 4 hours with Rapalog . Data Information : Error bars indicate standard deviation . Statistics performed by two - way ANOVA ; * * indicates a p - value < 0 . 01 , * * * indicates a p - value < 0 . 001 . ns indicates not significant . Over one thousand cells quantified per condition . Experiments repeated at least 3 times .

O O O O O O O O O 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 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

Representative images of WT or WIPI2 ko HeLa cells expressing EGFP-Nix1-188-FKBP (green), mtKeima, and FRB-Fis1T with or without 4 hours of Rapalog immunostained for endogenous Tom20 (red) and GABARAP (white). Scale bar: 50µm. Automated puncta counting of GABARAP in WT or WIPI2 ko cells normalized to cell number in control conditions or after 4 hours with Rapalog. Data Information: Error bars indicate standard deviation. Statistics performed by two-way ANOVA; ** indicates a p-value<0.01, *** indicates a p-value<0.001. ns indicates not significant. Over one thousand cells quantified per condition. Experiments repeated at least 3 times.


GFP pulldown of EGFP - Nix1 - 188 - FKBP in WT , WIPI2 ko , or FIP200 ko cells after 4 hours of Rapalog treatment . FIP200 cropped due to the appearance of non - specific bands after IP . Data Information : Error bars indicate standard deviation . Statistics performed by two - way ANOVA ; * * indicates a p - value < 0 . 01 , * * * indicates a p - value < 0 . 001 . ns indicates not significant . Over one thousand cells quantified per condition . Experiments repeated at least 3 times .

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GFP pulldown of EGFP-Nix1-188-FKBP in WT, WIPI2 ko, or FIP200 ko cells after 4 hours of Rapalog treatment. FIP200 cropped due to the appearance of non-specific bands after IP. Data Information: Error bars indicate standard deviation. Statistics performed by two-way ANOVA; ** indicates a p-value<0.01, *** indicates a p-value<0.001. ns indicates not significant. Over one thousand cells quantified per condition. Experiments repeated at least 3 times.


Representative images of endogenous Halo - WIPI2 in U2OS cells and stained with JF646 Halo dye ( magenta ) expressing mtKeima ( cyan ) , FRB - Fis1T , and FKBP - EGFP - Nix1 - 87 , FKBP - EGFP - Nix60 - 188 , or FKBP - EGFP - Nix1 - 70 ( green ) after 16 hours of Rapalog treatment . Data Information : Error bars indicate standard deviation between wells . Over one thousand cells quantified per condition . Statistics performed by two - way ANOVA ; * indicates a p - value < 0 . 05 , * * indicates a p - value < 0 . 01 , * * * * indicates a p - value < 0 . 0001 . ns indicates not significant . Experiments repeated at least 3 times .

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Representative images of endogenous Halo -WIPI2 in U2OS cells and stained with JF646 Halo dye (magenta) expressing mtKeima (cyan), FRB-Fis1T, and FKBP-EGFP-Nix1-87, FKBP-EGFP-Nix60-188, or FKBP-EGFP-Nix1-70 (green) after 16 hours of Rapalog treatment. Data Information: Error bars indicate standard deviation between wells. Over one thousand cells quantified per condition. Statistics performed by two-way ANOVA;* indicates a p-value<0.05, ** indicates a p-value<0.01, **** indicates a p-value<0.0001. ns indicates not significant. Experiments repeated at least 3 times.


Automated puncta counting of endogenous Halo - WIPI2 in U2OS cells expressing either FKBP - EGFP - Nix1 - 87 ( LIR and MER ) , Nix60 - 188 ( MER ) , or Nix1 - 70 ( LIR ) after 16 hours of Rapalog treatment then fixed . Data Information : Error bars indicate standard deviation between wells . Over one thousand cells quantified per condition . Statistics performed by two - way ANOVA ; * indicates a p - value < 0 . 05 , * * indicates a p - value < 0 . 01 , * * * * indicates a p - value < 0 . 0001 . ns indicates not significant . Experiments repeated at least 3 times .

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Automated puncta counting of endogenous Halo -WIPI2 in U2OS cells expressing either FKBP-EGFP-Nix1-87 (LIR and MER), Nix60-188 (MER), or Nix1-70 (LIR) after 16 hours of Rapalog treatment then fixed. Data Information: Error bars indicate standard deviation between wells. Over one thousand cells quantified per condition. Statistics performed by two-way ANOVA;* indicates a p-value<0.05, ** indicates a p-value<0.01, **** indicates a p-value<0.0001. ns indicates not significant. Experiments repeated at least 3 times.


Percent cells above threshold line shown in panel A as indication of mitophagy measured by mtKeima after one week of stably expressing EGFP , or EGFP - Nix as WT or with W36A / L39A or L75A mutations . Data Information : Error bars indicate standard deviation between wells . Over one thousand cells quantified per condition . Statistics : Panel B - two - tailed unpaired student ' s t - test * * indicates a p - value < 0 . 01 , * * * * indicates a p - value < 0 . 0001 . ns indicates not significant . Experiments repeated at least 3 times .

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Percent cells above threshold line shown in panel A as indication of mitophagy measured by mtKeima after one week of stably expressing EGFP, or EGFP-Nix as WT or with W36A/L39A or L75A mutations. Data Information: Error bars indicate standard deviation between wells. Over one thousand cells quantified per condition. Statistics: Panel B - two-tailed unpaired student's t-test ** indicates a p-value<0.01, **** indicates a p-value<0.0001. ns indicates not significant. Experiments repeated at least 3 times.


Automated puncta counting of endogenous Halo - WIPI2 in U2OS cells expressing FKBP - EGFP - Nix1 - 188 as WT or with W36A / L39 ( LIR ) or L75A ( MER ) mutations after 16 hours of Rapalog treatment then fixed . Data Information : Error bars indicate standard deviation between wells . Over one thousand cells quantified per condition . Statistics : panels C two - way ANOVA ; * * indicates a p - value < 0 . 01 , * * * * indicates a p - value < 0 . 0001 . ns indicates not significant . Experiments repeated at least 3 times .

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Automated puncta counting of endogenous Halo -WIPI2 in U2OS cells expressing FKBP-EGFP-Nix1-188 as WT or with W36A/L39 (LIR) or L75A (MER) mutations after 16 hours of Rapalog treatment then fixed. Data Information: Error bars indicate standard deviation between wells. Over one thousand cells quantified per condition. Statistics: panels C two-way ANOVA; ** indicates a p-value<0.01, **** indicates a p-value<0.0001. ns indicates not significant. Experiments repeated at least 3 times.


GFP pulldown of EGFP alone , EGFP - Nix1 - 188 - FKBP as WT , W36A / L39A , or L75A mutants after 4 hours of Rapalog treatment .

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GFP pulldown of EGFP alone, EGFP-Nix1-188-FKBP as WT, W36A/L39A, or L75A mutants after 4 hours of Rapalog treatment.


GFP pulldown of FKBP - EGFP - WIPI2 as WT or with R108E / R125E mutations after 24 hours of Phenanthroline treatment .

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GFP pulldown of FKBP-EGFP-WIPI2 as WT or with R108E/R125E mutations after 24 hours of Phenanthroline treatment.


Representative images of colon tumors were shown . Scale bars , 10 mm . Tumor number , tumor size and tumor load in each mouse was measured .

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Representative images of colon tumors were shown. Scale bars, 10 mm. Tumor number, tumor size and tumor load in each mouse was measured.


Histological analysis of colon tumors was shown by hematoxylin and eosin ( HE ) staining . Tumors were microscopically analyzed and classified as low or high grade . Histological score was assessed by a pathologist . Scale bars , 25 µm .

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Histological analysis of colon tumors was shown by hematoxylin and eosin (HE) staining. Tumors were microscopically analyzed and classified as low or high grade. Histological score was assessed by a pathologist. Scale bars, 25 µm.