( A ) The GRFs of Neil3 recognizes PAR . The recombinant GST fusion proteins were incubated with PAR and the protein - PAR complex was pulled down by glutathione agarose beads . Samples were serially diluted three - fold , spotted onto the nitrocellulose membrane , and subjected to dot blotting using anti - PAR antibody ( top panel ) . FL represents full length Neil3 , N - ter represents N - terminal Neil3 ( 1 - 300 aa ) , Mid represents middle Neil3 ( 300 - 404 aa ) , and GRFs represents C - terminal Neil3 GRFs domain ( 480 - 606 aa ) . Recombinant GST protein was used as the negative control ( NC ) . The GST fusion proteins were also examined by the SDS - PAGE followed with Coomassie blue staining ( middle panel ) . The relative PAR - binding in the pull down assay was summarized in the graph ( bottom panel ) . Values are mean ± SD of three assays .

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(A) The GRFs of Neil3 recognizes PAR. The recombinant GST fusion proteins were incubated with PAR and the protein-PAR complex was pulled down by glutathione agarose beads. Samples were serially diluted three-fold, spotted onto the nitrocellulose membrane, and subjected to dot blotting using anti-PAR antibody (top panel). FL represents full length Neil3, N-ter represents N-terminal Neil3 (1-300 aa), Mid represents middle Neil3 (300-404 aa), and GRFs represents C-terminal Neil3 GRFs domain (480-606 aa). Recombinant GST protein was used as the negative control (NC). The GST fusion proteins were also examined by the SDS-PAGE followed with Coomassie blue staining (middle panel). The relative PAR-binding in the pull down assay was summarized in the graph (bottom panel). Values are mean ± SD of three assays.


( B ) Tandem co - immunoprecipitation and Western blotting assays demonstrate that Neil3 interacts with ADP - ribosylated H2AX but not the E141A mutant H2AX .

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(B) Tandem co-immunoprecipitation and Western blotting assays demonstrate that Neil3 interacts with ADP-ribosylated H2AX but not the E141A mutant H2AX.


( C , D ) The GRFs of Neil3 is sufficient and required for the interaction with ADP - ribosylated H2AX . Tandem co - immunoprecipitation and Western blotting assays were

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(C,D) The GRFs of Neil3 is sufficient and required for the interaction with ADP-ribosylated H2AX. Tandem co-immunoprecipitation and Western blotting assays were used


( E , F ) The relocation kinetic of full - length Neil3 or the GRFs of Neil3 to DNA lesions was analyzed . GFP - tagged full length Neil3 or the GRFs of Neil3 was expressed in the indicated cells , and relocation kinetics was monitored in a time course following laser microirradiation ( left panel ) . The laser stripe is indicated with yellow arrowheads . The relocation kinetics is shown in the right panel . Data represent mean ± SD from three biologically independent experiments ( right panel ) . At least 20 cells were included in each experiment .

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(E,F) The relocation kinetic of full-length Neil3 or the GRFs of Neil3 to DNA lesions was analyzed. GFP-tagged full length Neil3 or the GRFs of Neil3 was expressed in the indicated cells, and relocation kinetics was monitored in a time course following laser microirradiation (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represent mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.


( G , H ) The recruitment of full - length Neil3 or the GRFs of Neil3 to the KR - induced oxidative lesions is mediated by H2AX ADP - ribosylation ( left panel ) . Scale bars , 2 μm . The foci were indicated with white arrowheads . Scale bars , 0 . 5 μm . Right panel : the percentage of Neil3 or GRFs Neil3 foci co - localized with KR was quantified . Mean value with SD is from 20 cells .

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(G,H) The recruitment of full-length Neil3 or the GRFs of Neil3 to the KR-induced oxidative lesions is mediated by H2AX ADP-ribosylation (left panel). Scale bars, 2 μm. The foci were indicated with white arrowheads. Scale bars, 0.5 μm. Right panel: the percentage of Neil3 or GRFs Neil3 foci co-localized with KR was quantified. Mean value with SD is from 20 cells.


( A ) PARP inhibition facilitates γH2AX following oxidative stress . The cells were pre - treated with olaparib ( 1 µM , 1 hour ) prior to H2O2 stimulation . 0 . 1 % DMSO in medium for 1 hour as mock . γH2AX or H2AX was detected using Western blotting and immunostaining assays . The intensity of γH2AX levels were examined by Western blotting normalized by β - actin and averaged . Values are mean ± SD of three assays . P - values were calculated using Student ' s t - test . * * P < 0 . 01 ( left panel ) . The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8 .

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(A) PARP inhibition facilitates γH2AX following oxidative stress. The cells were pre-treated with olaparib (1 µM, 1 hour) prior to H2O2 stimulation. 0.1 % DMSO in medium for 1 hour as mock. γH2AX or H2AX was detected using Western blotting and immunostaining assays. The intensity of γH2AX levels were examined by Western blotting normalized by β-actin and averaged. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. **P < 0.01 (left panel). The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8.


( B ) The effect of H2AX E141 ADP - ribosylation on γH2AX following oxidative stress . γH2AX or H2AX was detected using Western blotting and immunostaining assays . The intensity of γH2AX levels were examined by Western blotting normalized by β - actin and averaged . Values are mean ± SD of three assays . P - values were calculated using Student ' s t - test . * * P < 0 . 01 ( left panel ) . The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8 .

(B) The effect of H2AX E141 ADP-ribosylation on γH2AX following oxidative stress. γH2AX or H2AX was detected using Western blotting and immunostaining assays. The intensity of γH2AX levels were examined by Western blotting normalized by β-actin and averaged. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. **P < 0.01 (left panel). The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8.


( C ) The intensity of γH2AX is increased in cell expressing the E141A mutant following KillerRed - induced oxidative DNA lesions ( left panel ) . Scale bar , 2 μm . The foci were indicated with white arrowheads . Scale bar , 0 . 5 μm . Right panel : the relative intensity of γH2AX at the site of tetR ( intensity of γH2AX at the site of tetR / intensity of γH2AX area away from tetR ) was quantified . Mean value with SD is the relative intensity of γH2AX in 20 cells .

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(C) The intensity of γH2AX is increased in cell expressing the E141A mutant following KillerRed-induced oxidative DNA lesions (left panel). Scale bar, 2 μm. The foci were indicated with white arrowheads. Scale bar, 0.5 μm. Right panel: the relative intensity of γH2AX at the site of tetR (intensity of γH2AX at the site of tetR/intensity of γH2AX area away from tetR) was quantified. Mean value with SD is the relative intensity of γH2AX in 20 cells.


( D ) The level of MDC1 at KillerRed - induced oxidative DNA lesions is increased in the cells expressing the E141A mutant ( left panel ) . Scale bar , 2 μm . The foci were indicated with white arrowheads . Right panel : the percentage of MDC1 foci co - localized with KR was quantified . Mean value with SD is from 20 cells .

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(D) The level of MDC1 at KillerRed-induced oxidative DNA lesions is increased in the cells expressing the E141A mutant (left panel). Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: the percentage of MDC1 foci co-localized with KR was quantified. Mean value with SD is from 20 cells.


( E ) The recruitment of Neil3 / MDC1 chimera but not wild type Neil3 to the KR - induced oxidative lesions in cells expressing the E141A mutant ( left panel ) . Neil3 / MDC1 chimera : replaced the GRFs of Neil3 with the tandem BRCT domains of MDC1 . Scale bar , 2 μm . The foci were indicated with white arrowheads . Right panel : The percentage of detected protein foci co - localized with KR was quantified . Mean value with SD is from 20 cells .

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(E) The recruitment of Neil3/MDC1 chimera but not wild type Neil3 to the KR-induced oxidative lesions in cells expressing the E141A mutant (left panel). Neil3/MDC1 chimera: replaced the GRFs of Neil3 with the tandem BRCT domains of MDC1. Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: The percentage of detected protein foci co-localized with KR was quantified. Mean value with SD is from 20 cells.


( F ) AP sites were measured in cells expressing the E141A mutant or the Neil3 / MDC1 chimera , according to the standard curve . Values are mean ± SD of three assays .

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(F) AP sites were measured in cells expressing the E141A mutant or the Neil3/MDC1 chimera, according to the standard curve. Values are mean ± SD of three assays.


( G ) DNA replication of U2OS cells expression with individual wild type ( WT ) , the H2AX KO + WT , the E141A mutant ( E141A ) or the Neil3 / MDC1 Chimera ( Chimera ) were analyzed by BrdU incorporation following treatment with H2O2 , MMS or mock ( PBS ) . Represented images are shown in the left panel . The graph shows percentages of BrdU - positive cells expressing WT , E141A or Chimera ( right panel ) .

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(G) DNA replication of U2OS cells expression with individual wild type (WT), the H2AX KO + WT, the E141A mutant (E141A) or the Neil3/MDC1 Chimera (Chimera) were analyzed by BrdU incorporation following treatment with H2O2, MMS or mock (PBS). Represented images are shown in the left panel. The graph shows percentages of BrdU-positive cells expressing WT, E141A or Chimera (right panel).


A . Representative immunofluorescence images and quantifications showing that 3 months old Hnf1apKO mice have increased number of KI67 + ( red ) acinar cell nuclei co - staining with DAPI ( blue ) and Amylase ( green ) . Arrows point to KI67 + acinar cells in Hnf1apKO mouse . Scale bar is 200 μm . Acinar proliferation is represented as the average of the KI67 + / Amylase + cell ratio . Quantifications were performed on 3 random fields from 3 Pdx1Cre and 3 Hnf1apKO mice . P - values are from two - tailed Student ' s t test .

A. Representative immunofluorescence images and quantifications showing that 3 months old Hnf1apKO mice have increased number of KI67+ (red) acinar cell nuclei co-staining with DAPI (blue) and Amylase (green). Arrows point to KI67+ acinar cells in Hnf1apKO mouse. Scale bar is 200 μm. Acinar proliferation is represented as the average of the KI67+/Amylase+ cell ratio. Quantifications were performed on 3 random fields from 3 Pdx1Cre and 3 Hnf1apKO mice. P-values are from two-tailed Student's t test.


Representative H & E stainings of pancreata from KrasG12D and Hnf1apKO ; KrasG12D mice . B - D . KrasG12D and Hnf1apKO ; KrasG12D mice have normal morphology at 7 days . E - J . At 21 days Hnf1apKO ; KrasG12D mice show acinar - to - ductal metaplasia ( dashed encircled areas ) and regions with desmoplastic reaction ( asterisk ) , which are not observed in KrasG12D mice ( E , H ) . K - P . At 8 weeks KrasG12D pancreas show occasional abnormal ductal structures ( dashed encircled areas in N which is a magnification of squared dotted box in K ) and Hnf1apKO ; KrasG12D mice ( L , M , O , P ) present mucinous tubular complexes ( black arrows ) , and more advanced PanINs with luminal budding ( open arrows ) including foci of spindle cell proliferation ( asterisks ) and incipient infiltrative growth ( black dashed box area in O ) . Data information : Black dashed boxes in ( E , F , K , L and O ) indicate magnified areas in ( H , G , N , M and P ) respectively . Scale bars indicate 100 µm ( C , E , F , K , L ) , 50 µm ( O ) and 20 µm ( B , D , G , H - J , M , N , P ) .

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Representative H&E stainings of pancreata from KrasG12D and Hnf1apKO;KrasG12D mice. B-D. KrasG12D and Hnf1apKO;KrasG12D mice have normal morphology at 7 days. E-J. At 21 days Hnf1apKO;KrasG12D mice show acinar-to-ductal metaplasia (dashed encircled areas) and regions with desmoplastic reaction (asterisk), which are not observed in KrasG12D mice (E, H). K-P. At 8 weeks KrasG12D pancreas show occasional abnormal ductal structures (dashed encircled areas in N which is a magnification of squared dotted box in K) and Hnf1apKO;KrasG12D mice (L,M,O,P) present mucinous tubular complexes (black arrows), and more advanced PanINs with luminal budding (open arrows) including foci of spindle cell proliferation (asterisks) and incipient infiltrative growth (black dashed box area in O). Data information: Black dashed boxes in (E,F, K, L and O) indicate magnified areas in (H, G, N, M and P) respectively. Scale bars indicate 100 µm (C,E,F,K,L), 50 µm (O) and 20 µm (B,D,G,H-J,M,N,P).


A . Fold change ( FC ) in transcripts in Hnf1aaKO vs . control pancreas , plotted against significance ( - Log10 q ; genes significant at q < 0 . 05 are shown as colored dots above the horizontal line ) .

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A. Fold change (FC) in transcripts in Hnf1aaKO vs. control pancreas, plotted against significance (-Log10 q; genes significant at q<0.05 are shown as colored dots above the horizontal line).


B . GSEA showing that genes specific to differentiated acinar cells were downregulated in Hnf1aaKO pancreas , but not genes specific to islets or duct cells . Upregulated genes were enriched in genes specific to mesenchymal cells . Lineage - enriched genes were obtained from Muraro et al . ( Muraro et al . , 2016 ) .

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B. GSEA showing that genes specific to differentiated acinar cells were downregulated in Hnf1aaKO pancreas, but not genes specific to islets or duct cells. Upregulated genes were enriched in genes specific to mesenchymal cells. Lineage-enriched genes were obtained from Muraro et al. (Muraro et al., 2016).


D . GSEA revealed that Hnf1aaKO pancreas showed increased transcripts involved in oncogenic pathways such as EMT , MAPK , KRAS , PI3K - AKT .

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D. GSEA revealed that Hnf1aaKO pancreas showed increased transcripts involved in oncogenic pathways such as EMT, MAPK, KRAS, PI3K-AKT.


E . HNF1A promotes transcriptional activation of direct target genes . Left : HNF1A bound - genes were enriched among genes that showed downregulation in Hnf1a mutants , but not among upregulated genes . P - values and odds ratios ( O . R . ) calculated by Fisher ' s exact test . Right : Venn diagrams showing overlap of HNF1A - bound genes with genes that were downregulated and upregulated in Hnf1a mutant pancreas .

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E. HNF1A promotes transcriptional activation of direct target genes. Left: HNF1A bound-genes were enriched among genes that showed downregulation in Hnf1a mutants, but not among upregulated genes. P-values and odds ratios (O.R.) calculated by Fisher's exact test. Right: Venn diagrams showing overlap of HNF1A-bound genes with genes that were downregulated and upregulated in Hnf1a mutant pancreas.


A . Human orthologs of genes that were up - and down - regulated in Hnf1aaKO pancreas were also up - and down - regulated in human pancreas with low vs . high HNF1A expression ( lowest vs . highest expression deciles , respectively ) . A random list of 717 genes controlled for similar expression levels was used for comparison . Violin plots include median and interquartile ranges . Dots are average values for each gene . Kruskall Wallis P < 0 . 0001 .

A. Human orthologs of genes that were up- and down-regulated in Hnf1aaKO pancreas were also up- and down-regulated in human pancreas with low vs. high HNF1A expression (lowest vs. highest expression deciles, respectively). A random list of 717 genes controlled for similar expression levels was used for comparison. Violin plots include median and interquartile ranges. Dots are average values for each gene. Kruskall Wallis P<0.0001.


D . HNF1A mRNA levels differed in HNF1A LoF and control groups ( Kruskal - Wallis , P < 0 . 01 ) , despite considerable variability and overlap between groups . E . KDM6A mRNA levels were downregulated in HNF1A LoF tumors ( Kruskal - Wallis , P < 0 . 001 ) . Data information : Box plots in ( D and E ) show HNF1A and KDM6A expression in HNF1A LoF tumors ( n = 26 ) and Control 1 ( n = 39 ) and Control 2 ( n = 57 ) tumors . The horizontal central line marks the median . Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1 . 5 x interquartile range outside box limits .

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D. HNF1A mRNA levels differed in HNF1A LoF and control groups (Kruskal-Wallis, P<0.01), despite considerable variability and overlap between groups. E. KDM6A mRNA levels were downregulated in HNF1A LoF tumors (Kruskal-Wallis, P<0.001).Data information: Box plots in (D and E) show HNF1A and KDM6A expression in HNF1A LoF tumors (n=26) and Control 1 (n=39) and Control 2 (n=57) tumors. The horizontal central line marks the median. Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1.5 x interquartile range outside box limits.


H & E staining of pancreata from Kdm6apKO ; KrasG12D and KrasG12D mice . A - C . 7 day - old KrasG12D and Kdm6apKO ; KrasG12D mice showed normal morphology . D - I . At 21 days , KrasG12D mice showed normal pancreas morphology ( dotted area at in D is shown at large magnification in G ) whereas Kdm6apKO ; KrasG12D mice already show acinar - to - ductal metaplasia ( dashed encircled area in F ) , spindle cell proliferation ( asterisks in E and H ) , sarcomatoid architecture ( I ) and desmoplastic reaction ( black arrowhead in H ) . J - O . KrasG12D mice present occasional acinar - to - ductal metaplasia and low grade PanINs at 8 weeks ( M is a magnification of squared dotted box in J and see Fig 1N ) whereas at the same age pancreas from Kdm6apKO ; KrasG12D mice show massive remodeling ( K ) , extensive acinar - to - ductal metaplasia ( L and dashed encircled area in N ) , cancer with prominent spindle / mesenchymal proliferation , infiltrative growth ( black arrows and asterisk respectively in O ) and abundant stroma ( black arrowheads in N ) . Data information : Scale bars : 100 µm ( B , D , E , J , K ) or 20 µm ( A , C , F - I , L - N ) .

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H&E staining of pancreata from Kdm6apKO;KrasG12D and KrasG12D mice. A-C. 7 day-old KrasG12D and Kdm6apKO;KrasG12D mice showed normal morphology. D-I. At 21 days, KrasG12D mice showed normal pancreas morphology (dotted area at in D is shown at large magnification in G) whereas Kdm6apKO;KrasG12D mice already show acinar-to-ductal metaplasia (dashed encircled area in F), spindle cell proliferation (asterisks in E and H), sarcomatoid architecture (I) and desmoplastic reaction (black arrowhead in H). J-O. KrasG12D mice present occasional acinar-to-ductal metaplasia and low grade PanINs at 8 weeks (M is a magnification of squared dotted box in J and see Fig 1N) whereas at the same age pancreas from Kdm6apKO;KrasG12D mice show massive remodeling (K), extensive acinar-to-ductal metaplasia (L and dashed encircled area in N), cancer with prominent spindle/mesenchymal proliferation, infiltrative growth (black arrows and asterisk respectively in O) and abundant stroma (black arrowheads in N). Data information: Scale bars: 100 µm (B,D,E,J,K) or 20 µm (A,C,F-I, L-N).


A . Fold change ( FC ) and significance for transcripts in Kdm6apKO vs . control pancreas . Genes significant at q < 0 . 05 are shown as colored dots above the horizontal line .

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A. Fold change (FC) and significance for transcripts in Kdm6apKO vs. control pancreas. Genes significant at q<0.05 are shown as colored dots above the horizontal line.


B . GSEA showing that genes specific to differentiated acinar cells were downregulated in KDM6A - deficient pancreas , but not genes specific to islets or duct cells . Upregulated genes were enriched in genes specific to mesenchymal cells . Lineage - enriched genes were obtained from ( Muraro et al . , 2016 ) .

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B. GSEA showing that genes specific to differentiated acinar cells were downregulated in KDM6A-deficient pancreas, but not genes specific to islets or duct cells. Upregulated genes were enriched in genes specific to mesenchymal cells. Lineage-enriched genes were obtained from (Muraro et al., 2016).


D . GSEA showed increased expression of indicated oncogenic pathway genes in Kdm6apKO pancreas .

D. GSEA showed increased expression of indicated oncogenic pathway genes in Kdm6apKO pancreas.


E . KDM6A functions as a transcriptional activator of direct target genes in pancreatic cells . Left : Downregulated , but not upregulated , genes in Kdm6apKO pancreas were enriched for KDM6A binding . P - values and odds ratios ( O . R . ) were calculated by Fisher ' s exact test . Right : Venn diagrams showing overlap of down and upregulated genes with KDM6A binding .

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E. KDM6A functions as a transcriptional activator of direct target genes in pancreatic cells. Left: Downregulated, but not upregulated, genes in Kdm6apKO pancreas were enriched for KDM6A binding. P-values and odds ratios (O.R.) were calculated by Fisher's exact test. Right: Venn diagrams showing overlap of down and upregulated genes with KDM6A binding.


F . KDM6A - bound enhancers and promoters showed increased H3K27me3 and decreased H3K27ac in Kdm6apKO pancreas .

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F. KDM6A-bound enhancers and promoters showed increased H3K27me3 and decreased H3K27ac in Kdm6apKO pancreas.


G . Genes that were downregulated in Kdm6apKO pancreas ( KDM6A - dependent genes ) showed greatest changes in histone marks . Box plots show fold - changes of H3K27me3 ( left ) and H3K27ac ( right ) signals in Kdm6apKO compared to control . Signals were analyzed in KDM6A bound regions in promoters and enhancers of genes that were KDM6A dependent ( grey ; n = 420 regions ) and independent ( white ; n = 8035 regions ) . The signals are average values from ChIP - seq experiments in two biological replicates . The horizontal central line marks the median . Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1 . 5 x IQR outside box limits . P - values were determined by two - tailed Mann - Whitney U test .

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G. Genes that were downregulated in Kdm6apKO pancreas (KDM6A-dependent genes) showed greatest changes in histone marks. Box plots show fold-changes of H3K27me3 (left) and H3K27ac (right) signals in Kdm6apKO compared to control. Signals were analyzed in KDM6A bound regions in promoters and enhancers of genes that were KDM6A dependent (grey; n=420 regions) and independent (white; n=8035 regions). The signals are average values from ChIP-seq experiments in two biological replicates. The horizontal central line marks the median. Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1.5 x IQR outside box limits. P-values were determined by two-tailed Mann-Whitney U test.


H . Changes in KDM6A , H3K27me3 and H3K27ac ChIP - seq profiles in the Ppp4r4 - Serpina10 locus in Kdm6apKO pancreas .

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H. Changes in KDM6A, H3K27me3 and H3K27ac ChIP-seq profiles in the Ppp4r4-Serpina10 locus in Kdm6apKO pancreas.


I . qPCR of H3K27me3 , H3K27ac and H3K4me1 changes in promoter and enhancer regions highlighted in ( H ) ( R1 , R2 , R3 ) , and Serpin10 mRNA . Error bars show + / - SD and P - values are from two - tailed Student ' s t test , n = 3 .

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I. qPCR of H3K27me3, H3K27ac and H3K4me1 changes in promoter and enhancer regions highlighted in (H) (R1, R2, R3), and Serpin10 mRNA. Error bars show +/-SD and P-values are from two-tailed Student's t test, n=3.


J . KDM6A - bound genes that showed decreased expression in Kdm6apKO pancreas ( functional KDM6A binding sites ) showed simultaneous gain of H3K27me3 and loss and of H3K27ac .

O O O O 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

J. KDM6A-bound genes that showed decreased expression in Kdm6apKO pancreas (functional KDM6A binding sites) showed simultaneous gain of H3K27me3 and loss and of H3K27ac.


B . Co - binding analysis in functional KDM6A - bound enhancer and promoter regions revealed that HNF1A was the most enriched co - bound TF amongst three other acinar cell TFs . Binding regions of TAL1 in a non - pancreatic cell type and random binding sites were used as negative controls . P - values was determined by Fisher ' s exact test for peak comparisons using all enhancer and promoter regions as background .

B. Co-binding analysis in functional KDM6A-bound enhancer and promoter regions revealed that HNF1A was the most enriched co-bound TF amongst three other acinar cell TFs. Binding regions of TAL1 in a non-pancreatic cell type and random binding sites were used as negative controls. P-values was determined by Fisher's exact test for peak comparisons using all enhancer and promoter regions as background.


C . The most downregulated genes in Kdm6apKO pancreas are shown ranked by q - value , and are almost invariably bound by HNF1A and down regulated in Hnf1aaKO pancreas , or known to be direct HNF1A - dependent target genes from other studies ( red and purple , respectively ) .

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C. The most downregulated genes in Kdm6apKO pancreas are shown ranked by q-value, and are almost invariably bound by HNF1A and down regulated in Hnf1aaKO pancreas, or known to be direct HNF1A-dependent target genes from other studies (red and purple, respectively).


D , E . GSEA analysis on the Hnf1aaKO and Kdm6apKO ranked - ordered gene lists vs . their reciprocal up - or downregulated gene sets , demonstrated that KDM6A and HNF1A regulate similar genes .

D,E. GSEA analysis on the Hnf1aaKO and Kdm6apKO ranked-ordered gene lists vs. their reciprocal up- or downregulated gene sets, demonstrated that KDM6A and HNF1A regulate similar genes.


F . Expression changes in Hnf1aaKO and Kdm6apKO pancreas , showing that genes bound by KDM6A and downregulated in Kdm6apKO pancreas ( red dots ) were generally downregulated in Hnf1aaKO pancreas .

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F. Expression changes in Hnf1aaKO and Kdm6apKO pancreas, showing that genes bound by KDM6A and downregulated in Kdm6apKO pancreas (red dots) were generally downregulated in Hnf1aaKO pancreas.


G . HNF1A and KDM6A co - occupy the same regions in Pah , which is downregulated in Hnf1a and Kdm6a knock - out pancreas .

G. HNF1A and KDM6A co-occupy the same regions in Pah, which is downregulated in Hnf1a and Kdm6a knock-out pancreas.


H . Genes that were co - bound by KDM6A and HNF1A showed greatest downregulation in Kdm6apKO pancreas , compared with KDM6A - bound genes that were not bound by HNF1A . Box plots show median and IQR of Log2 TPM fold - changes and whiskers extend to highest and lowest data points within 1 . 5 x IQR outside box limits . P - values were determined by two - tailed Student ' s t tests and n = 4 replicates per condition .

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H. Genes that were co-bound by KDM6A and HNF1A showed greatest downregulation in Kdm6apKO pancreas, compared with KDM6A-bound genes that were not bound by HNF1A. Box plots show median and IQR of Log2 TPM fold-changes and whiskers extend to highest and lowest data points within 1.5 x IQR outside box limits. P-values were determined by two-tailed Student's t tests and n=4 replicates per condition.


A . Co - immunoprecipitation of endogenous HNF1A and KDM6A followed by western blot demonstrated that HNF1A is in the same complex as KDM6A .

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A. Co-immunoprecipitation of endogenous HNF1A and KDM6A followed by western blot demonstrated that HNF1A is in the same complex as KDM6A.


B . Western blot showing loss of HNF1A and unchanged KDM6A in Hnf1a - / - pancreas .

B. Western blot showing loss of HNF1A and unchanged KDM6A in Hnf1a-/- pancreas.


C . Differential binding analysis of KDM6A in Hnf1a - / - vs . wild type pancreas . Pink dots below zero ( 1873 sites ) show regions with reduced KDM6A binding and pink dots above zero ( 118 sites ) are regions with increased binding at FDR < 0 . 05 .

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C. Differential binding analysis of KDM6A in Hnf1a-/- vs. wild type pancreas. Pink dots below zero (1873 sites) show regions with reduced KDM6A binding and pink dots above zero (118 sites) are regions with increased binding at FDR<0.05.


Regions that show reduced KDM6A binding in Hnf1a - / - chromatin are strongly bound by HNF1A and are highly enriched in HNF1 motifs . P - values in ( D ) were calculated with two - tailed Mann - Whitney U

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Regions that show reduced KDM6A binding in Hnf1a-/- chromatin are strongly bound by HNF1A and are highly enriched in HNF1 motifs. P-values in (D) were calculated with two-tailed Mann-Whitney U test


Regions that show reduced KDM6A binding in Hnf1a - / - chromatin are strongly bound by HNF1A and are highly enriched in HNF1 motifs . P - values in ( E ) with Fisher ' s exact test .

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Regions that show reduced KDM6A binding in Hnf1a-/- chromatin are strongly bound by HNF1A and are highly enriched in HNF1 motifs. P-values in (E) with Fisher's exact test.


F . KDM6A binding is markedly reduced in HNF1A and KDM6A co - bound regions in Hnf1a - / - pancreas , but not in other KDM6A bound regions .

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F. KDM6A binding is markedly reduced in HNF1A and KDM6A co-bound regions in Hnf1a-/- pancreas, but not in other KDM6A bound regions.


G . Genes that loose KDM6A binding in Hnf1a - mutant pancreas are predominantly downregulated in Hnf1aaKO pancreas and are direct HNF1A target genes ( red dots ) .

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G. Genes that loose KDM6A binding in Hnf1a-mutant pancreas are predominantly downregulated in Hnf1aaKO pancreas and are direct HNF1A target genes (red dots).


B . The ER marker ER - tdTomato ( magenta ) is co - localized with ectopically expressed Wrb - EGFP ( green ) in inner saccular HCs of 5 - dpf zebrafishlarvae ( ' control ' refers to either wild - type or + / pwi fish that were picked in a phenotypic screen and did not exert any abnormal behavioral phenotype ) . While the left HC solely expressed ER - tdTomato , the neighboring HC expressed both , ER - tdTomato and Wrb - GFP . Colocalization between both proteins occurs in areas exhibiting white pixels . Scale bars : 5

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B. The ER marker ER-tdTomato (magenta) is co-localized with ectopically expressed Wrb-EGFP (green) in inner saccular HCs of 5-dpf zebrafishlarvae ('control' refers to either wild-type or +/pwi fish that were picked in a phenotypic screen and did not exert any abnormal behavioral phenotype). While the left HC solely expressed ER-tdTomato, the neighboring HC expressed both, ER-tdTomato and Wrb-GFP. Colocalization between both proteins occurs in areas exhibiting white pixels. Scale bars: 5 µm


C . Projection of confocal sections of control inner ear HCs immunolabelled for otoferlin ( magenta ) and expressing an EGFP - tagged truncated Wrb fragment containing only the cytosolic coiled - coiled domains ( Wrbcc - EGFP , green ) . Wrbcc - EGFP distribution was diffuse and found throughout the HC .

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C. Projection of confocal sections of control inner ear HCs immunolabelled for otoferlin (magenta) and expressing an EGFP-tagged truncated Wrb fragment containing only the cytosolic coiled-coiled domains (Wrbcc-EGFP, green). Wrbcc-EGFP distribution was diffuse and found throughout the HC.


D . Projection of confocal sections of inner ear HCs of 5 - dpf control ( D ) and Wrb - deficient pwi mutant fish ( wrb pwi / pwi ; D ' ) , showing strongly reduced otoferlin immunofluorescence in the mutant HCs . The color look - up table used represents higher pixel intensities with warmer colors .

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D. Projection of confocal sections of inner ear HCs of 5-dpf control (D) and Wrb-deficient pwi mutant fish (wrb pwi/pwi; D'), showing strongly reduced otoferlin immunofluorescence in the mutant HCs. The color look-up table used represents higher pixel intensities with warmer colors.


E . A representative transgenically - rescued pwi - mutant HC ( white arrow ) , expressing Wrb - GFP , exhibits a strongly increased otoferlin signal ( magenta ) in direct comparison to the neighboring non - rescued mutant HCs . ( E ' ) Same image as in E but intensity - coded for otoferlin fluorescence .

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E. A representative transgenically-rescued pwi-mutant HC (white arrow), expressing Wrb-GFP, exhibits a strongly increased otoferlin signal (magenta) in direct comparison to the neighboring non-rescued mutant HCs. (E') Same image as in E but intensity-coded for otoferlin fluorescence.


F . A representative control HC expressing Wrbcc - EGFP ( white arrow ) , immunolabelled for otoferlin ( magenta ) . The Wrbcc - EGFP - expressing HC shows significantly less otoferlin ( F ' ) , suggesting a dominant negative effect of Wrbcc in otoferlin biogenesis . The transfected HC is encircled with a dashed line .

F. A representative control HC expressing Wrbcc-EGFP (white arrow), immunolabelled for otoferlin (magenta). The Wrbcc-EGFP-expressing HC shows significantly less otoferlin (F'), suggesting a dominant negative effect of Wrbcc in otoferlin biogenesis. The transfected HC is encircled with a dashed line.


G . Quantification otoferlin downregulation of Wrbcc - EGFP shown in F . Otoferlin immunofluorescence intensity of Wrbcc - EGFP transfected inner ear HCs n = 22 , from three 5 - dpf control larva ) showed significant decrease of ~ 43 % compared to the adjacent Wrbcc - EGFP negative wild - type HCs ( n = 22 , from the same larva ) . Fluorescent intensity values were normalized with the average value of Wrbcc - EGFP negative HCs . * * p < 0 .

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G. Quantification otoferlin downregulation of Wrbcc-EGFP shown in F. Otoferlin immunofluorescence intensity of Wrbcc-EGFP transfected inner ear HCs n = 22, from three 5-dpf control larva) showed significant decrease of ~43% compared to the adjacent Wrbcc-EGFP negative wild-type HCs (n = 22, from the same larva). Fluorescent intensity values were normalized with the average value of Wrbcc-EGFP negative HCs. **p < 0.01


H . Quantification of otoferlin immunohistochemistry data shown in ( D ) from hair cells of control ( n = 88 HCs from three 5 - dpf larva ) , wrb pwi / pwi ( n = 80 HCs from 3 sibling larva ) normalized against the mean intensity value of the control group and Wrb - GFP transfected wrb pwi / pwi zebrafish inner ears . Mutant HCs showed ~ 82 % reduction in otoferlin fluorescence intensity when compared with control HCs imaged under the same conditions . Otoferlin fluorescent intensity of Wrb - EGFP transfected mutant HCs ( n = 24 , from three , 5 - dpf larva ) showed significant increases compared to adjacent Wrb - EGFP negative mutant HCs ( n = 24 from the same larva ) . Fluorescent intensity values were normalized with the average value of Wrb - EGFP negative HCs . * * * p < 0 .

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H. Quantification of otoferlin immunohistochemistry data shown in (D) from hair cells of control (n = 88 HCs from three 5-dpf larva), wrb pwi/pwi (n = 80 HCs from 3 sibling larva) normalized against the mean intensity value of the control group and Wrb-GFP transfected wrb pwi/pwi zebrafish inner ears. Mutant HCs showed ~82% reduction in otoferlin fluorescence intensity when compared with control HCs imaged under the same conditions. Otoferlin fluorescent intensity of Wrb-EGFP transfected mutant HCs (n = 24, from three, 5-dpf larva) showed significant increases compared to adjacent Wrb-EGFP negative mutant HCs (n = 24 from the same larva). Fluorescent intensity values were normalized with the average value of Wrb-EGFP negative HCs. ***p<0.001


I - I ' . Acoustic startle reflex measurements of ( I ) live , intact , 5 - dpf control ( n = 20 ) and wrb pwi / pwi ( n = 18 ) zebrafishlarva , where almost 100 % of the larva responded to five successive acoustic stimuli at 3 seconds intervals . In wrb pwi / pwi animals , only 39 % of larva responded to the first stimulus , thereafter , the number of responding larva decreased rapidly to less than 6 % . ( I ' ) Acoustic startle reflexes of 5 - dpf zebrafish larva after injection with either EGFP capped mRNA ( control , n = 16 , or wrb pwi / pwi n = 17 ) , Wrb - EGFP capped mRNA ( control , n = 20 , or wrb pwi / pwi n = 20 ) or two different concentrations of otoferlin mRNA ( 1x : control , n = 13 ; wrb pwi / pwi n = 11 ; 2x : control , n = 16 ; wrb pwi / pwi n = 6 ) . In contrast to EGFP mRNA injection , Wrb - EGFP as well as otoferlin mRNAs could partially rescue the acoustic startle reflex in the mutants , while not displaying detrimental effects on overall zebrafish morphology and development . Please note the dose - dependent effect of otoferlin mRNA injection . Similar results as in 1x EGFP mRNA were also obtained from injection of 2X EGFP mRNA as control ( data not shown ) . Measurements from two ( 2x otoferlin ) or three ( all other ) different experiments were compiled in the graph .

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

I-I'. Acoustic startle reflex measurements of (I) live, intact, 5-dpf control (n = 20) and wrb pwi/pwi (n = 18) zebrafishlarva, where almost 100% of the larva responded to five successive acoustic stimuli at 3 seconds intervals. In wrb pwi/pwi animals, only 39% of larva responded to the first stimulus, thereafter, the number of responding larva decreased rapidly to less than 6%. (I') Acoustic startle reflexes of 5-dpf zebrafish larva after injection with either EGFP capped mRNA (control, n = 16, or wrb pwi/pwi n = 17), Wrb-EGFP capped mRNA (control, n = 20, or wrb pwi/pwi n = 20) or two different concentrations of otoferlin mRNA (1x: control, n = 13; wrb pwi/pwi n = 11; 2x: control, n = 16; wrb pwi/pwi n = 6). In contrast to EGFP mRNA injection, Wrb-EGFP as well as otoferlin mRNAs could partially rescue the acoustic startle reflex in the mutants, while not displaying detrimental effects on overall zebrafish morphology and development. Please note the dose-dependent effect of otoferlin mRNA injection. Similar results as in 1x EGFP mRNA were also obtained from injection of 2X EGFP mRNA as control (data not shown). Measurements from two (2x otoferlin) or three (all other) different experiments were compiled in the graph.


A . HZZ - OTOFop , carrying a C - terminal glycosylation site ( opsin tag ) was purified alone or in complex with wild - type or an ATPase - deficient mutant version of TRC40 and incubated in the absence or presence of ER - derived rough microsomes ( RM ) . Membrane integration ( glycosylation ) was monitored by SDS - PAGE and immunoblot using an anti - opsin antibody . Where indicated , EndoH was used to remove N - linked oligosaccharides .

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A. HZZ-OTOFop, carrying a C-terminal glycosylation site (opsin tag) was purified alone or in complex with wild-type or an ATPase-deficient mutant version of TRC40 and incubated in the absence or presence of ER-derived rough microsomes (RM). Membrane integration (glycosylation) was monitored by SDS-PAGE and immunoblot using an anti-opsin antibody. Where indicated, EndoH was used to remove N-linked oligosaccharides.


B . HZZ - OTOFop in complex with wild - type or mutant TRC40 was incubated with RM in the presence or absence of ATP and membrane insertion was monitored by opsin - specific immunoblot .

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B. HZZ-OTOFop in complex with wild-type or mutant TRC40 was incubated with RM in the presence or absence of ATP and membrane insertion was monitored by opsin-specific immunoblot.


C . HZZ - OTOFop in complex with wild - type TRC40 was incubated in the presence of RM , trypsin - treated rough microsomes ( TRM ) or in the presence of WRBcc or CAMLcyt . Membrane insertion was monitored by opsin - specific immunoblot .

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C. HZZ-OTOFop in complex with wild-type TRC40 was incubated in the presence of RM, trypsin-treated rough microsomes (TRM) or in the presence of WRBcc or CAMLcyt. Membrane insertion was monitored by opsin-specific immunoblot.


D . Quantification of relative protein glycosylation shown in B ( n = 3 ) . Data are represented as mean ± SEM . TRC40gr , mutated version of TRC40 ; * * p < 0 . 01 ; * * * p < 0 . 001 ( Student ' s two - sample t - test ) .

D. Quantification of relative protein glycosylation shown in B (n = 3). Data are represented as mean ± SEM. TRC40gr, mutated version of TRC40; **p<0.01; ***p<0.001 (Student's two-sample t-test).


E . Quantification of relative protein glycosylation of the data shown in C ( n = 3 ) . Data are represented as mean ± SEM . * * * p < 0 . 001 ( Student ' s two - sample t - test )

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E. Quantification of relative protein glycosylation of the data shown in C (n = 3). Data are represented as mean ± SEM. ***p<0.001 (Student's two-sample t-test)


A . Grand averages of auditory brainstem responses ( ABR ) from Wrbfl / fl : CreA ( red traces ; SEM pink ) and Wrb + / + : CreA ( black traces ; SEM grey ) mice . ABRs were recorded for three separate age groups ( A - A ' ' , as indicated in the graph ) using click stimulation at 80 dB ( peak equivalent ) and 20 Hz stimulation rate . There was an age - progressive reduction of ABR amplitude in Wrbfl / fl : CreA mice .

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A. Grand averages of auditory brainstem responses (ABR) from Wrbfl/fl:CreA (red traces; SEM pink) and Wrb+/+:CreA (black traces; SEM grey) mice. ABRs were recorded for three separate age groups (A-A'', as indicated in the graph) using click stimulation at 80 dB (peak equivalent) and 20 Hz stimulation rate. There was an age- progressive reduction of ABR amplitude in Wrbfl/fl:CreA mice.


B . ABRs from Wrbfl / fl : CreA mice showed a progressive threshold increase , Wrb + / + : CreA mice and Wrbfl / fl mice lacking Cre recombinase had normal thresholds . Statistical comparison of the threshold at 12 kHz was done by Kruskal - Wallis test , p values are from post - hoc Dunn ' s multiple comparison test . Measurements in which no ABR was observed at the maximal available tone burst level ( 90 dB ) or click level ( 120 dB ) scored as 100 dB threshold . Data are represented as mean ± SEM .

B. ABRs from Wrbfl/fl:CreA mice showed a progressive threshold increase, Wrb+/+:CreA mice and Wrbfl/fl mice lacking Cre recombinase had normal thresholds. Statistical comparison of the threshold at 12 kHz was done by Kruskal-Wallis test, p values are from post-hoc Dunn's multiple comparison test. Measurements in which no ABR was observed at the maximal available tone burst level (90 dB) or click level (120 dB) scored as 100 dB threshold. Data are represented as mean ± SEM.


A - D . Representative maximum projections of confocal sections of ( A - B ) P14 and ( C - D ) P20 Wrb + / + : CreA and Wrbfl / fl : CreA apical turn organs of Corti following immunolabeling against otoferlin ( green ) and Vglut3 ( magenta ) processed and imaged under identical conditions . A ' - D ' show individual otoferlin stainings with an intensity - coded lookup table . A ' ' - D ' ' present single xz projections at a central point through a representative IHC to illustrate otoferlin subcellular distribution . Scale bars in A - D ' represent 10 μm , in A ' ' - D ' ' 2 µm . Note the dramatic reduction in otoferlin fluorescence and the alteration in its distribution as well as a change in cell shape and position of nuclei ( for detailed analysis of cell shape and nuclei position , please refer to Figure EV5 ) .

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A-D. Representative maximum projections of confocal sections of (A-B) P14 and (C-D) P20 Wrb+/+:CreA and Wrbfl/fl:CreA apical turn organs of Corti following immunolabeling against otoferlin (green) and Vglut3 (magenta) processed and imaged under identical conditions. A'-D' show individual otoferlin stainings with an intensity-coded lookup table. A''-D'' present single xz projections at a central point through a representative IHC to illustrate otoferlin subcellular distribution. Scale bars in A-D' represent 10 μm, in A''-D'' 2 µm. Note the dramatic reduction in otoferlin fluorescence and the alteration in its distribution as well as a change in cell shape and position of nuclei (for detailed analysis of cell shape and nuclei position, please refer to Figure EV5).


E - G . Semiquantitative analysis of otoferlin immunofluorescence in the apical parts of IHCs from Wrbfl / fl : CreA mice using averaged regions of interests in cell ' s maximal projections ( outlined with a dotted line in G for a single representative IHC ) normalized to the maximum values observed in the respective control IHCs . Averaged supra nuclear coronal line profiles as illustrated with a dashed line in G are shown for ( F ) P14 - 16 and ( F ' ) P18 - 21 IHCs . * * * p < 0 . 001 versus Wrb + / + :

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E-G. Semiquantitative analysis of otoferlin immunofluorescence in the apical parts of IHCs from Wrbfl/fl:CreA mice using averaged regions of interests in cell's maximal projections (outlined with a dotted line in G for a single representative IHC) normalized to the maximum values observed in the respective control IHCs. Averaged supra nuclear coronal line profiles as illustrated with a dashed line in G are shown for (F) P14-16 and (F') P18-21 IHCs. ***p<0.001 versus Wrb+/+:CreA


H - J . Semiquantitative analysis of otoferlin immunofluorescence in the basal parts of IHCs from Wrbfl / fl : CreA mice using averaged regions of interests in cell ' s maximal projections ( outlined with a dotted line in J for a single representative IHC ) normalized to the maximum values observed in the respective control IHCs . Averaged supranuclear coronal line profiles as illustrated with a dashed line in G are shown for ( I ) P14 - 16 and ( I ' ) P18 - 21 IHCs . * * * p < 0 . 001 versus Wrb + / + :

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H-J. Semiquantitative analysis of otoferlin immunofluorescence in the basal parts of IHCs from Wrbfl/fl:CreA mice using averaged regions of interests in cell's maximal projections (outlined with a dotted line in J for a single representative IHC) normalized to the maximum values observed in the respective control IHCs. Averaged supranuclear coronal line profiles as illustrated with a dashed line in G are shown for (I) P14-16 and (I') P18-21 IHCs. ***p<0.001 versus Wrb+/+:CreA


A . Representative electron micrograph of a Wrb + / + : CreA IHC ribbon synapse . Scale bar : 120 nm .

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A. Representative electron micrograph of a Wrb+/+:CreA IHC ribbon synapse. Scale bar: 120 nm.


B - B ' . In Wrbfl / fl : CreA ( depicted are two consecutive ultrathin sections ) IHCs , accumulations of large partially amorphous vesicles were observed close to ribbon synapses ( red arrows ) , but also further away in the cytoplasm ( cyan arrows ) . Moreover , cisternal structures resembling ( r ) ER are found unusually close to the ribbon ( orange arrow in B ' ) . Scale bar : 120 nm .

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B-B'. In Wrbfl/fl:CreA (depicted are two consecutive ultrathin sections) IHCs, accumulations of large partially amorphous vesicles were observed close to ribbon synapses (red arrows), but also further away in the cytoplasm (cyan arrows). Moreover, cisternal structures resembling (r)ER are found unusually close to the ribbon (orange arrow in B'). Scale bar: 120 nm.


E . Quantification in random ultrathin sections ( Wrb + / + : CreA n = 28 , from two animals ; Wrbfl / fl : CreA n = 44 , from three animals ) of the numbers of membrane - proximal " MP " vesicles ( in close proximity to membrane and ribbon ) ; vesicles within a 100 nm range along the active zone membrane ; vesicles at the lower " proximal " and the upper " distal " half of the ribbon as well as vesicle > 70 nm ( " large vesicles " ) in a radius of 800 nm around the ribbon . The vesicle number was significantly reduced in Wrbfl / fl : CreA IHCs at both ribbon parts but not at the presynaptic membrane . F . Average vesicle diameters of the membrane - proximal vesicles and the vesicles at both halves of the ribbon . The mean vesicle diameter increases significantly at both parts of the ribbon in Wrbfl / fl : CreA IHCs . Data are represented as mean ± SEM . Student ' s two - sample t - test * * * p < 0 .

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E. Quantification in random ultrathin sections (Wrb+/+:CreA n = 28, from two animals; Wrbfl/fl:CreA n = 44, from three animals) of the numbers of membrane-proximal "MP" vesicles (in close proximity to membrane and ribbon); vesicles within a 100 nm range along the active zone membrane; vesicles at the lower "proximal" and the upper "distal" half of the ribbon as well as vesicle >70 nm ("large vesicles") in a radius of 800 nm around the ribbon. The vesicle number was significantly reduced in Wrbfl/fl:CreA IHCs at both ribbon parts but not at the presynaptic membrane.F. Average vesicle diameters of the membrane-proximal vesicles and the vesicles at both halves of the ribbon. The mean vesicle diameter increases significantly at both parts of the ribbon in Wrbfl/fl:CreA IHCs. Data are represented as mean ± SEM. Student's two-sample t-test ***p<0.001


A . Ca2 + current - voltage relationship of P14 - P17 IHCs of Wrbfl / fl : CreA ( n = 17 IHCs ) and Wrb + / + : CreA ( n = 15 IHCs ) mice in 2 mM extracellular [ Ca2 + ] showed no change in amplitude or voltage - dependence of the Ca2 + current . Data are represented as mean ± SEM .

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A. Ca2+ current-voltage relationship of P14-P17 IHCs of Wrbfl/fl:CreA (n = 17 IHCs) and Wrb+/+:CreA (n = 15 IHCs) mice in 2 mM extracellular [Ca2+] showed no change in amplitude or voltage-dependence of the Ca2+ current. Data are represented as mean ± SEM.


B . Representative Ca2 + current ( top ) and Cm changes ( bottom ) in Wrbfl / fl : CreA and Wrb + / + : CreA IHCs in response to a 20 - ms step depolarizations to - 14 mV ( the potential eliciting the maximum Ca2 + current ) .

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B. Representative Ca2+ current (top) and Cm changes (bottom) in Wrbfl/fl:CreA and Wrb+/+:CreA IHCs in response to a 20-ms step depolarizations to -14 mV (the potential eliciting the maximum Ca2+ current).


C . Representative Ca2 + current ( top ) and Cm changes ( bottom ; finite impulse response filtered ) in Wrbfl / fl : CreA and Wrb + / + : CreA IHCs in response to a 100 ms step depolarizations to - 14 mV .

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C. Representative Ca2+ current (top) and Cm changes (bottom; finite impulse response filtered) in Wrbfl/fl:CreA and Wrb+/+:CreA IHCs in response to a 100 ms step depolarizations to -14 mV.


D . Exocytic ΔCm ( top ) and corresponding Ca2 + current integrals , QCa ( bottom ) for various depolarization durations . Data are grand averages of the cells ' means . Exocytic ΔCm was reduced in the knockout from 20 ms onwards , indicating reduced sustained exocytosis . Data from OtofPga / Pga animals were replotted for direct comparison from ( Pangrsic et al , 2010 ) .

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D. Exocytic ΔCm (top) and corresponding Ca2+ current integrals, QCa (bottom) for various depolarization durations. Data are grand averages of the cells' means. Exocytic ΔCm was reduced in the knockout from 20 ms onwards, indicating reduced sustained exocytosis. Data from OtofPga/Pga animals were replotted for direct comparison from (Pangrsic et al, 2010).


E . ΔCm / QCa2 + ratio indicates a lower efficacy of Ca2 + influx in driving exocytosis in Wrbfl / fl : CreA IHCs , for stimuli of 20 ms or longer . Data represents means ± SEM ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 .

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E. ΔCm/QCa2+ ratio indicates a lower efficacy of Ca2+ influx in driving exocytosis in Wrbfl/fl:CreA IHCs, for stimuli of 20 ms or longer. Data represents means ± SEM; *p<0.05; **p<0.01; ***p<0.001


A . All SGNs of Wrbfl / fl : CreA mice had spontaneous firing rates ( SR ) below 20 Hz ( p > 0 . 05 , Kolmogorow - Smirnov test ) .

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A. All SGNs of Wrbfl/fl:CreA mice had spontaneous firing rates (SR) below 20 Hz (p>0.05, Kolmogorow-Smirnov test).


B . Representative tuning curves from Wrbfl / fl : CreA and Wrb + / + : CreA SGNs indicating preserved active cochlear amplification despite disruption of Wrb in IHCs .

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B. Representative tuning curves from Wrbfl/fl:CreA and Wrb+/+:CreA SGNs indicating preserved active cochlear amplification despite disruption of Wrb in IHCs.


C . Normal thresholds at the characteristic frequency ( CF ) in Wrbfl / fl : CreA SGNs .

C. Normal thresholds at the characteristic frequency (CF) in Wrbfl/fl:CreA SGNs.


D - D ' ' . Mean poststimulus time histograms ( PSTH ) ± SEM of Wrbfl / fl : CreA and Wrb + / + : CreA SGNs in response to 50 ms tone bursts presented at CF , 30 dB above threshold at stimulus rates of 2 Hz ( D ) , 5 Hz ( D ' ) , or 10 Hz ( D ' ' ) . While the general response pattern was preserved , spike rates were drastically reduced in Wrbfl / fl : CreA SGNs , especially at higher stimulus rates . Data from auditory neurons ( SGN and cochlear nucleus ) of OtofPga / Pga animals were replotted for direct comparison from ( Pangrsic et al , 2010 ) .

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D-D''. Mean poststimulus time histograms (PSTH) ± SEM of Wrbfl/fl:CreA and Wrb+/+:CreA SGNs in response to 50 ms tone bursts presented at CF, 30 dB above threshold at stimulus rates of 2 Hz (D), 5 Hz (D'), or 10 Hz (D''). While the general response pattern was preserved, spike rates were drastically reduced in Wrbfl/fl:CreA SGNs, especially at higher stimulus rates. Data from auditory neurons (SGN and cochlear nucleus) of OtofPga/Pga animals were replotted for direct comparison from (Pangrsic et al, 2010).


E . Spike rates in response to sound onset ( maximum rate in PSTH with 0 . 5 ms binwidth ) and adapted rates ( averaged between 35 - 45 ms after stimulus onset ) were significantly lower in Wrbfl / fl : CreA SGNs ( stimulus rate 5 Hz ; p < 0 . 001 ) .

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E. Spike rates in response to sound onset (maximum rate in PSTH with 0.5 ms binwidth) and adapted rates (averaged between 35-45 ms after stimulus onset) were significantly lower in Wrbfl/fl:CreA SGNs (stimulus rate 5 Hz; p<0.001).


F . In line with the reduction in spike rates , first spike latency ( FSL ) following stimulus onset was greatly increased in Wrbfl / fl : CreA SGNs compared to Wrb + / + : CreA ( stimulus rate 5 Hz ; p < 0 . 001 ) .

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F. In line with the reduction in spike rates, first spike latency (FSL) following stimulus onset was greatly increased in Wrbfl/fl:CreA SGNs compared to Wrb+/+:CreA (stimulus rate 5 Hz; p<0.001).


G . Illustration of the stimulus paradigm for forward masking experiments : a 100 ms masker tone presented at CF , 30 dB above threshold was followed by a silent interval of variable duration and a 15 ms probe tone ( CF , 30 dB above threshold ) . Bottom : in Wrbfl / fl : CreA SGNs , the response to the first 5 ms of the masker probe tone tone ( shown as a fraction of the response to the first 5ms of the masker ) was strongly reduced , and the half time of recovery ( from normalized curves ) was increased from 39 . 2 ± 12 . 2 ms in Wrb + / + : CreA to 215 . 7 ± 51 . 2 ms in Wrbfl / fl : CreA SGNs ( p < 0 . 001 , Mann - Whitney U test ) . Data represent means ± SEM .

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G. Illustration of the stimulus paradigm for forward masking experiments: a 100 ms masker tone presented at CF, 30 dB above threshold was followed by a silent interval of variable duration and a 15 ms probe tone (CF, 30 dB above threshold). Bottom: in Wrbfl/fl:CreA SGNs, the response to the first 5 ms of the masker probe tone tone (shown as a fraction of the response to the first 5ms of the masker) was strongly reduced, and the half time of recovery (from normalized curves) was increased from 39.2 ± 12.2 ms in Wrb+/+:CreA to 215.7 ± 51.2 ms in Wrbfl/fl:CreA SGNs (p<0.001, Mann-Whitney U test). Data represent means ± SEM.


C . Apparent decrease in insertion efficiency for the TMD mutant version . While the WT otoferlin glycosylation amounted to 3 . 84 ± 0 . 53 , the TMD mutant had only 2 . 13 ± 0 . 65 ( n = 3 , p < 0 . 05 ) . Data are represented as mean ± SEM .

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C. Apparent decrease in insertion efficiency for the TMD mutant version. While the WT otoferlin glycosylation amounted to 3.84 ± 0.53, the TMD mutant had only 2.13 ± 0.65 (n=3, p<0.05). Data are represented as mean ± SEM.


A . Visual reinforcement audiometry performed in the free field : a mild hearing loss .

A. Visual reinforcement audiometry performed in the free field: a mild hearing loss.


B . Distortion product otoacoustic emissions ( DPOAEs ) were detected in each ear , whereasC . Auditory brainstem responses ( ABR ) were lacking , together providing the signature of auditory synaptopathy or neuropathy .

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B. Distortion product otoacoustic emissions (DPOAEs) were detected in each ear, whereasC. Auditory brainstem responses (ABR) were lacking, together providing the signature of auditory synaptopathy or neuropathy.


D - E . Cochlear microphonic potentials ( CM ) , summating potential ( SP ) and compound action potential ( CAP ) recorded through transtympanic electrocochleography ( ECochG ) in response to clicks at decreasing stimulus intensities are superimposed on the corresponding potentials recorded from one normally - hearing control ( range as measured at 120 dB 4 . 31 - 28 . 02 µV in 20 normally - hearing children ) . Means and standard errors of peak latency and amplitude are also reported for each potential category . The ECochG waveform resulting from CM cancellation in the control begins with an abrupt negative deflection , the SP , followed by a negative peak , the neural CAP . In the child with the OTOF TMD mutation , the ECochG responses begin with a rapid negative deflection that peaks at the same latency as the SP in the control and has comparable amplitude . This is followed by a low - amplitude negative potential that peaks at the same CAP in controls but shows a markedly prolonged duration . In all graphs time " 0 " refers to CM onset . R : right , L : left . F . Means and standard errors of peak latency and amplitude are reported for each potential category .

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D-E. Cochlear microphonic potentials (CM), summating potential (SP) and compound action potential (CAP) recorded through transtympanic electrocochleography (ECochG) in response to clicks at decreasing stimulus intensities are superimposed on the corresponding potentials recorded from one normally-hearing control (range as measured at 120 dB 4.31-28.02 µV in 20 normally-hearing children). Means and standard errors of peak latency and amplitude are also reported for each potential category. The ECochG waveform resulting from CM cancellation in the control begins with an abrupt negative deflection, the SP, followed by a negative peak, the neural CAP. In the child with the OTOF TMD mutation, the ECochG responses begin with a rapid negative deflection that peaks at the same latency as the SP in the control and has comparable amplitude. This is followed by a low-amplitude negative potential that peaks at the same CAP in controls but shows a markedly prolonged duration. In all graphs time "0" refers to CM onset. R: right, L: left.F. Means and standard errors of peak latency and amplitude are reported for each potential category.


A Immunostaining of a murine knee joint at 18 . 5 dpc with an anti GCP - 2 or IgG control antibody .

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A Immunostaining of a murine knee joint at 18.5 dpc with an anti GCP-2 or IgG control antibody.


B Immunohistochemistry for GCP - 2 ( red ) in the knee of a human 5 - month old fetus .

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B Immunohistochemistry for GCP-2 (red) in the knee of a human 5-month old fetus.


C , D Immunostaining for GCP - 2 in an adult ( 12 week old ) mouse knee ( C ) or in adult human articular cartilage ( D ) .

C, D Immunostaining for GCP-2 in an adult (12 week old) mouse knee (C) or in adult human articular cartilage (D).


GAG content as assessed by Alcian blue staining with densitometry ( A ) A HAC micromasses treated with a GCP - 2 neutralizing antibody ( n = 4 ) or IgG control ( n = 4 ) ; p values were determined by the Mann - Whitney U test .

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GAG content as assessed by Alcian blue staining with densitometry (A) A HAC micromasses treated with a GCP-2 neutralizing antibody (n=4) or IgG control (n=4); p values were determined by the Mann-Whitney U test.


GAG content as assessed by Alcian blue staining with spectrophotometric B C28 / I2 micromasses treated with GCP - 2 siRNA ( n = 12 ) or scrambled control siRNA ( Scr ; n = 14 ) ; p values were determined by unpaired two - tailed Student ' s t test .

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GAG content as assessed by Alcian blue staining with spectrophotometric B C28/I2 micromasses treated with GCP-2 siRNA (n=12) or scrambled control siRNA (Scr; n=14); p values were determined by unpaired two-tailed Student's t test.


GAG content as assessed by Alcian blue staining spectrophotometric C C3H10T½ micromasses treated for 3 days with recombinant GCP - 2 100 ng / ml ( n = 4 ) or vehicle ( n = 4 ) or BMP - 2 ( n = 4 ) ; p values were determined by ANOVA followed by Tukey HSD post hoc test .

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GAG content as assessed by Alcian blue staining spectrophotometric C C3H10T½ micromasses treated for 3 days with recombinant GCP-2 100 ng/ml (n=4) or vehicle (n=4) or BMP-2 (n=4); p values were determined by ANOVA followed by Tukey HSD post hoc test.


D qPCR for ACAN ( Aggrecan ) expression in C3H10T½ micromasses treated for 3 days with recombinant GCP - 2 ( 100 ng / ml ) ( n = 3 ) or vehicle ( n = 4 ) ; ACAN - Aggrecan ;

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D qPCR for ACAN (Aggrecan) expression in C3H10T½ micromasses treated for 3 days with recombinant GCP-2 (100 ng/ml) (n=3) or vehicle (n=4); ACAN - Aggrecan;


E spectrophotometric quantification of GAG release ( dimethylmethylene blue assay ) in supernatant of human cartilage explants treated with recombinant GCP - 2 ( n = 5 ) or vehicle ( n = 5 ) and incubated for

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E spectrophotometric quantification of GAG release (dimethylmethylene blue assay) in supernatant of human cartilage explants treated with recombinant GCP-2 (n=5) or vehicle (n=5) and incubated for 24h


F Toluidine blue staining of ectopic cartilage explants collected 2 weeks after subcutaneous co - implantation of HACs in a 10 : 1 ratio with growth - arrested COS7 expressing either GFP ( n = 3 ) or GCP - 2 ( n = 3 ) and quantification - on the right - hand side - of the metachromatically stained area as % of total area ; scale bar = 200µm ;

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F Toluidine blue staining of ectopic cartilage explants collected 2 weeks after subcutaneous co-implantation of HACs in a 10:1 ratio with growth-arrested COS7 expressing either GFP (n=3) or GCP-2 (n=3) and quantification - on the right-hand side - of the metachromatically stained area as % of total area; scale bar =200µm;


G Collagen 2 ( Col2 ) immunostaining ( left ) and quantification ( right ) as % of total area ; scale bar = 100µm ; n = 3 per group ; p values were determined by unpaired two - tailed Student ' s t test .

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G Collagen 2 (Col2) immunostaining (left) and quantification (right) as % of total area; scale bar = 100µm; n = 3 per group; p values were determined by unpaired two-tailed Student's t test.


A , B qPCR for ( A ) Runx2 and ( B ) Col1A1 expression in HAC micromasses treated for 24 hours with recombinant GCP - 2 ( 100 ng / ml ) or vehicle ( n = 4 ) ; Runx2 = Runt - related transcription factor 2 , Col1A1 = collagen type 1 alpha 1 ; n =

A, B qPCR for (A) Runx2 and (B) Col1A1 expression in HAC micromasses treated for 24 hours with recombinant GCP-2 (100 ng/ml) or vehicle (n=4); Runx2 = Runt-related transcription factor 2, Col1A1 = collagen type 1 alpha 1; n=4


C Alkaline phosphatase staining ( red ) in HAC monolayer after 3 week treatment with T3 hormone ( 100 ng / ml ) , T3 hormone + GCP - 2 ( 100ng / ml ) , or control medium ; scale bar 100µm .

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C Alkaline phosphatase staining (red) in HAC monolayer after 3 week treatment with T3 hormone (100 ng/ml), T3 hormone + GCP-2 (100ng/ml), or control medium; scale bar 100µm.


D quantification of alkaline phosphatase stained area ( n = 3 ) ; p values were determined by fitting a generalised linear model followed by pairwise comparison of the estimated marginal means .

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D quantification of alkaline phosphatase stained area (n=3); p values were determined by fitting a generalised linear model followed by pairwise comparison of the estimated marginal means.


E qPCR for Col10A1 ( n = 3 ) of HAC 3 weeks after treatment with vehicle , T3 ( 100 ng / ml ) or T3 + GCP - 2 ( 100 ng / ml ) ; n = 3 ;

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E qPCR for Col10A1 (n=3) of HAC 3 weeks after treatment with vehicle, T3 (100 ng/ml) or T3+GCP-2 (100 ng/ml); n=3;


F , H C3H10T½ monolayers were treated with osteogenic medium ( OM ) or OM + GCP - 2 ( 100 ng / ml ) : ( F ) number of cells per well positive for alkaline phosphatase staining ( n = 3 in OM and n = 4 in OM + GCP - 2 ) , ( G ) number of alkaline phosphatase positive nodules per well ( n = 7 ) , and ( H ) spectrophotometric quantification of alizarin red staining at 570nm ( n = 6 )

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F, H C3H10T½ monolayers were treated with osteogenic medium (OM) or OM + GCP-2 (100 ng/ml): (F) number of cells per well positive for alkaline phosphatase staining (n=3 in OM and n=4 in OM+GCP-2), (G) number of alkaline phosphatase positive nodules per well (n=7), and (H) spectrophotometric quantification of alizarin red staining at 570nm (n=6)


GAG binding capacity of wildtype GCP - 2 , and mutants ( K101E single mutant , K105E single mutant , GCP - 2 - D = K101E _ K105E double mutant and GCP - 2 - T = triple mutant K100E _ K101E _ K105E ) was assessed by ( B ) a heparin microtiter plate binding assay measuring the interaction of biotinylated - heparin ( b - heparin ) with immobilized

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GAG binding capacity of wildtype GCP-2, and mutants (K101E single mutant, K105E single mutant, GCP-2-D = K101E_K105E double mutant and GCP-2-T = triple mutant K100E_ K101E_K105E) was assessed by (B) a heparin microtiter plate binding assay measuring the interaction of biotinylated-heparin (b-heparin) with immobilized proteins


( C ) heparin affinity chromatography equilibrated and run in PBS , pH 7 . 4 . In ( C ) the proteins are eluted with a salt gradient from 0 - 2 M NaCl monitored by the conductivity ( Cond ) , where 20 and 90 mS / cm correspond to 193 and 1360 mM NaCl , respectively ; GCP - 2 - T , GCP - 2 - D , K101E , K105E and WT proteins elute at 530 mM , 617 mM , 815 mM , 850 mM and 1024 mM NaCl , respectively .

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(C) heparin affinity chromatography equilibrated and run in PBS, pH 7.4. In (C) the proteins are eluted with a salt gradient from 0-2 M NaCl monitored by the conductivity (Cond), where 20 and 90 mS/cm correspond to 193 and 1360 mM NaCl, respectively; GCP-2-T, GCP-2-D, K101E, K105E and WT proteins elute at 530 mM, 617 mM, 815 mM, 850 mM and 1024 mM NaCl, respectively.


D , E Maximum migratory activity of WT GCP - 2 ( 50nM ) was compared to GCP - 2 mutants ( 50nM ) in ( D ) chemotaxis and ( E ) trans - endothelial migration assays in CXCR2 - expressing 300 - 19 pre B cells . WT = wildtype GCP - 2 ; K101E = GCP - 2 _ K101E single mutant ; K105E = GCP - 2 _ K105E single mutant ; D = GCP - 2 _ K101E _ K105E double mutant ; T = GCP - 2 _ K100E _ K101E _ K105E triple mutant ; n = 4 ; p values were determined by fitting a generalised linear model followed by pairwise comparison of the estimated marginal means .

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D, E Maximum migratory activity of WT GCP-2 (50nM) was compared to GCP-2 mutants (50nM) in (D) chemotaxis and (E) trans-endothelial migration assays in CXCR2-expressing 300-19 pre B cells. WT = wildtype GCP-2; K101E = GCP-2_K101E single mutant; K105E = GCP-2_K105E single mutant; D = GCP-2_K101E_K105E double mutant; T = GCP-2_K100E_K101E_K105E triple mutant; n=4; p values were determined by fitting a generalised linear model followed by pairwise comparison of the estimated marginal means.


C28 / I2 micromasses stimulated with WT GCP - 2 , GCP - 2 triple mutant ( GCP - 2 - T ) or untreated and assessed for ( F ) AKT phosphorylation levels by western blotting ; V = vehicle treated samples , GCP - 2 = WT GCP - 2 treated samples , GCP - 2 - T = triple mutant GCP - 2 treated samples , pAKT = phosphorylated AKT , tAKT = total AKT . On the right , quantification of the density of the bands using ImageJ . Five independent experiments were analyzed . The data were scaled and p values were determined by fitting a generalised linear model followed by pairwise comparison of the estimated marginal means .

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

C28/I2 micromasses stimulated with WT GCP-2, GCP-2 triple mutant (GCP-2-T) or untreated and assessed for (F) AKT phosphorylation levels by western blotting; V = vehicle treated samples, GCP-2 = WT GCP-2 treated samples, GCP-2-T = triple mutant GCP-2 treated samples, pAKT = phosphorylated AKT, tAKT = total AKT. On the right, quantification of the density of the bands using ImageJ. Five independent experiments were analyzed. The data were scaled and p values were determined by fitting a generalised linear model followed by pairwise comparison of the estimated marginal means.


C28 / I2 micromasses stimulated with WT GCP - 2 , GCP - 2 triple mutant ( GCP - 2 - T ) or untreated and assessed G spectrophotometric quantification of proteoglycan content by Alcian blue staining . p values were determined by Kruskal - Wallis test with Dunn ' s post - hoc test ; ( n = 8 ) .

O O O O O O O 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

C28/I2 micromasses stimulated with WT GCP-2, GCP-2 triple mutant (GCP-2-T) or untreated and assessed G spectrophotometric quantification of proteoglycan content by Alcian blue staining. p values were determined by Kruskal-Wallis test with Dunn's post-hoc test; (n=8).


C28 / I2 micromasses stimulated with WT GCP - 2 , GCP - 2 triple mutant ( GCP - 2 - T ) or untreated and assessed H ACAN expression levels by qPCR ; ACAN = Aggrecan ; p values were determined by ANOVA with Tukey ' s HSD post - hoc test ; n = 4 .

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

C28/I2 micromasses stimulated with WT GCP-2, GCP-2 triple mutant (GCP-2-T) or untreated and assessed H ACAN expression levels by qPCR; ACAN = Aggrecan; p values were determined by ANOVA with Tukey's HSD post-hoc test; n=4.


I - J Real time quantitative PCR of COL2A1 ( I ) and COL10A1 ( J ) mRNA normalized to β actin . p values were determined by fitting a generalised linear model followed by comparison of the estimated marginal means ; n = 4 control , 5 GCP - 2 and GCP - 2 - T for COL2A1 and n = 6 per group for COL10A1 .

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

I-J Real time quantitative PCR of COL2A1 (I) and COL10A1 (J) mRNA normalized to β actin. p values were determined by fitting a generalised linear model followed by comparison of the estimated marginal means; n=4 control, 5 GCP-2 and GCP-2-T for COL2A1 and n=6 per group for COL10A1.


K Number of neutrophils counted within the intercondylar space on sections from mice killed 4 days after an intra - articular injection of adenovirus encoding GFP ( n = 3 ) , GCP - 2 ( n = 3 ) and GCP - 2 - T ( n = 3 ) ; p values were determined by fitting a generalised linear model ( family = poisson ) followed by pairwise comparison of the estimated marginal means .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Number of neutrophils counted within the intercondylar space on sections from mice killed 4 days after an intra-articular injection of adenovirus encoding GFP (n= 3), GCP-2 (n=3) and GCP-2-T (n=3); p values were determined by fitting a generalised linear model (family=poisson) followed by pairwise comparison of the estimated marginal means.


B Weekly pain measurement by incapacitance , shown as the percentage of bodyweight loaded on the operated leg in GFP ( n = 15 ) vs GCP - 2 ( n = 14 ) and GFP vs GCP - 2 - T ( n = 15 ) treated mice . Circles show the mean , and error bars show 95 % confidence intervals .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Weekly pain measurement by incapacitance, shown as the percentage of bodyweight loaded on the operated leg in GFP (n= 15) vs GCP-2 (n=14) and GFP vs GCP-2-T (n=15) treated mice. Circles show the mean, and error bars show 95% confidence intervals.


C Incapacitance at week 10 ( final time - point ) ; red line indicates 50 % loading ( no pain ) ; p values were determined by unpaired , two - tailed one sample Student ' s t test testing the hypothesis that mice loaded 50 % body weight on the operated limb ( n = 13 in the GFP group and n = 14 in the GCP - 2 and GCP - 2 - T groups ) . D The area under the curve ( AUC ) of incapacitance calculated starting from week 6 ; p values were determined by ANOVA with Tukey ' s HSD post - hoc test ( n = 13 in the GFP group and 14 in the GCP - 2 and GCP - 2 - T group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Incapacitance at week 10 (final time-point); red line indicates 50% loading (no pain); p values were determined by unpaired, two-tailed one sample Student's t test testing the hypothesis that mice loaded 50% body weight on the operated limb (n=13 in the GFP group and n=14 in the GCP-2 and GCP-2-T groups). D The area under the curve (AUC) of incapacitance calculated starting from week 6; p values were determined by ANOVA with Tukey's HSD post-hoc test (n=13 in the GFP group and 14 in the GCP-2 and GCP-2-T group).


E , F Osteoarthritis severity assessed using ( E ) OARSI scoring system 10 weeks after MLI ; GFP ( n = 7 ) , GCP - 2 ( n = 8 ) and GCP - 2 - T ( n = 7 ) ; p values were determined by fitting a generalized linear model followed by comparison of the estimated marginal means . F representative image ( Safranin O staining ) for each treatment ; arrows indicate the tidemark ;

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Osteoarthritis severity assessed using (E) OARSI scoring system 10 weeks after MLI; GFP (n= 7), GCP-2 (n=8) and GCP-2-T (n=7); p values were determined by fitting a generalized linear model followed by comparison of the estimated marginal means. F representative image (Safranin O staining) for each treatment; arrows indicate the tidemark;


G Quantification of osteophytes area ; GFP ( n = 7 ) , GCP - 2 ( n = 7 ) and GCP - 2 - T ( n = 8 ) ; H Representative images with osteophyte highlighted I Osteophyte maturity score ( n = 7 ) ; p values were determined by fitting a generalized linear model followed by pairwise comparison of the estimated marginal means .

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

G Quantification of osteophytes area; GFP (n= 7), GCP-2 (n=7) and GCP-2-T (n=8); H Representative images with osteophyte highlighted I Osteophyte maturity score (n=7); p values were determined by fitting a generalized linear model followed by pairwise comparison of the estimated marginal means.


( a ) Ba / F3 cells expressing myrPI ( 3 ) K - ER , myrPKB - ER , FOXO3 ( A3 ) - ER or FOXO4 ( A3 ) - ER were stimulated with 4 - OHT for 4 h ( myrPI ( 3 ) K - ER , myrPKB - ER ) or 8 h ( FOXO3 ( A3 ) - ER and FOXO4 ( A3 ) - ER ) and microarray analyses were performed . Shown are the fold changes relative to control cells for glutamine synthetase ( GS ) , Mxi1 and Pink1 . Data are represented as mean values of one experiment performed in quadruplicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Ba/F3 cells expressing myrPI(3)K-ER, myrPKB-ER, FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT for 4 h (myrPI(3)K-ER, myrPKB-ER) or 8 h (FOXO3(A3)-ER and FOXO4(A3)-ER) and microarray analyses were performed. Shown are the fold changes relative to control cells for glutamine synthetase (GS), Mxi1 and Pink1. Data are represented as mean values of one experiment performed in quadruplicate.


( b ) Ba / F3 cells expressing either myrPI ( 3 ) K - ER or myrPKB - ER were cytokine starved overnight and stimulated with 4 - OHT . Relative mRNA levels of glutamine synthetase were analysed using quantitative rtPCR . Data are represented as mean ± s . e . m . normalized for B2M ( n = 4 ) . * P0 . 05 , * * P0 . 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-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

(b) Ba/F3 cells expressing either myrPI(3)K-ER or myrPKB-ER were cytokine starved overnight and stimulated with 4-OHT. Relative mRNA levels of glutamine synthetase were analysed using quantitative rtPCR. Data are represented as mean ± s.e.m. normalized for B2M (n=4). *P0.05, **P0.01.


( c ) Ba / F3 cells expressing either myrPI ( 3 ) K - ER or myrPKB - ER were cytokine starved overnight and stimulated with 4 - OHT . Cell lysates were analysed for protein levels of phospho - FOXO3 ( T32 ) , glutamine synthetase , p27 and actin . Shown are representative blots ( n = 4 ) .

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

(c) Ba/F3 cells expressing either myrPI(3)K-ER or myrPKB-ER were cytokine starved overnight and stimulated with 4-OHT. Cell lysates were analysed for protein levels of phospho-FOXO3 (T32), glutamine synthetase, p27 and actin. Shown are representative blots (n=4).


( d ) Ba / F3 cells expressing either FOXO3 ( A3 ) - ER or FOXO4 ( A3 ) - ER were stimulated with 4 - OHT in the presence of mIL - 3 . Relative mRNA levels of glutamine synthetase were analysed using quantitative rtPCR . Data are represented as mean ± s . e . m . values normalized for B2M ( n = 3 ) . * * P0 . 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 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

(d) Ba/F3 cells expressing either FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Relative mRNA levels of glutamine synthetase were analysed using quantitative rtPCR. Data are represented as mean ±  s.e.m. values normalized for B2M (n=3). **P0.01.


( e ) Ba / F3 cells expressing either FOXO3 ( A3 ) - ER or FOXO4 ( A3 ) - ER were stimulated with 4 - OHT in the presence of mIL - 3 . Cell lysates were analysed for protein levels of glutamine synthetase , p27 and actin . Shown are representative blots ( n = 3 ) .

O 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

(e) Ba/F3 cells expressing either FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=3).


( f ) Wild - type Ba / F3 cells were cytokine starved overnight and stimulated with mIL - 3 . Cell lysates were analysed for protein levels of phospho - FOXO3 ( T32 ) , glutamine synthetase , p27 and actin . Shown are representative blots ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wild-type Ba/F3 cells were cytokine starved overnight and stimulated with mIL-3. Cell lysates were analysed for protein levels of phospho-FOXO3 (T32), glutamine synthetase, p27 and actin. Shown are representative blots (n=4).


( g ) Wild - type Ba / F3 cells were incubated with LY294002 in the presence of mIL - 3 . Cell lysates were analysed for protein levels of glutamine synthetase , p27 and actin . Shown are representative blots ( n = 3 ) .

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

(g) Wild-type Ba/F3 cells were incubated with LY294002 in the presence of mIL-3. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=3).


( h ) FOXO1 , 3 , 4 − / − MEFs and wild - type MEFs were incubated with LY294002 ( 10 μM ) for the indicated times . Cell RNA was isolated and relative mRNA levels of glutamine synthetase were analysed using quantitative PCR . Data are represented as mean values normalized for B2M ( n = 2 ) . Uncropped images of blots are shown in Supplementary Fig . S7 .

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

(h) FOXO1,3,4−/− MEFs and wild-type MEFs were incubated with LY294002 (10 μM) for the indicated times. Cell RNA was isolated and relative mRNA levels of glutamine synthetase were analysed using quantitative PCR. Data are represented as mean values normalized for B2M (n=2). Uncropped images of blots are shown in Supplementary Fig. S7.


( a ) Ba / F3 cells expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT and actinomycin D ( 1 μg ml − 1 ) or dimethylsulphoxide ( DMSO ) as a control in the presence of mIL - 3 for 16 h . Cell lysates were analysed for protein levels of glutamine synthetase ( GS ) , p27 and actin . Shown are representative blots ( n = 3 ) .

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 B-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

(a) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and actinomycin D (1 μg ml−1) or dimethylsulphoxide (DMSO) as a control in the presence of mIL-3 for 16 h. Cell lysates were analysed for protein levels of glutamine synthetase (GS), p27 and actin. Shown are representative blots (n=3).


( b ) Glutamine synthetase reporter plasmids carrying mutations in putative FOXO - binding sites were transfected in HEK293 cells together with Renilla and FOXO3 ( A3 ) as indicated . Luciferase activity was measured 40 h after transfection . Data are depicted as relative luciferase units ( RLU ) compared with the control . Shown are mean ± s . e . m . values ( n = 3 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Glutamine synthetase reporter plasmids carrying mutations in putative FOXO-binding sites were transfected in HEK293 cells together with Renilla and FOXO3(A3) as indicated. Luciferase activity was measured 40 h after transfection. Data are depicted as relative luciferase units (RLU) compared with the control. Shown are mean ± s.e.m. values (n=3)


( a ) DLD1 cells expressing FOXO3 ( A3 ) - ER were transiently transfected with GFP - ULK2 and stimulated with 4 - OHT and MSO for 24 h in glutamine - free DMEM containing 0 . 1 % FCS . Cells were stained for LC3 and analysed by confocal microscopy . Shown are representative pictures ( n = 3 ) . Scale bars , 20 μm .

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

(a) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP-ULK2 and stimulated with 4-OHT and MSO for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for LC3 and analysed by confocal microscopy. Shown are representative pictures (n=3). Scale bars, 20 μm.


( d ) DLD1 cells expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT . Cell lysates were analysed for protein levels of glutamine synthetase , p27 and actin . Shown are representative blots ( n = 4 ) .

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

(d) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=4).


( e ) The osteosarcoma cell line U2OS expressing FOXO ( A3 ) - ER was stimulated with 4 - OHT , cells were lysed and equal amounts of proteins were analysed for levels of glutamine synthetase and actin .

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

(e) The osteosarcoma cell line U2OS expressing FOXO(A3)-ER was stimulated with 4-OHT, cells were lysed and equal amounts of proteins were analysed for levels of glutamine synthetase and actin.


( f ) MSCs expressing FOXO ( A3 ) - ER were stimulated with 4 - OHT , cells were lysed and equal amounts of proteins were analysed for levels of glutamine synthetase and actin . Shown are representative blots ( n = 3 ) .

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

(f) MSCs expressing FOXO(A3)-ER were stimulated with 4-OHT, cells were lysed and equal amounts of proteins were analysed for levels of glutamine synthetase and actin. Shown are representative blots (n=3).


( g ) Wild - type , daf - 2 and daf - 16 mutant worms were synchronized to L1 by hypochlorite treatment and were placed on nematode growth medium agar plates . After five days worms were lysed in imidazole and analysed for glutamine synthetase activity .

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

(g) Wild-type, daf-2 and daf-16 mutant worms were synchronized to L1 by hypochlorite treatment and were placed on nematode growth medium agar plates. After five days worms were lysed in imidazole and analysed for glutamine synthetase activity.