Figure 5 ( A - E ) Optical density ( 600nm ) variations during in vitro growth assay of Salmonella enterica Typhimurium ( SL1344 ) co - cultured with stool - purified IgA1 ( A ) or IgA2 ( B ) from non - IBD ( n = 4 ) , CD ( n = 6 ) and UC ( n = 3 ) . Comparison between IgA1 and IgA2 co - culture in non - IBD ( C ) , CD ( D ) , and UC ( E ) . Two - way ANOVA with Holm - Sidak correction after D ' Agostino - Pearson nomality test , p - values are as follows : ( A ) UC curve : 2h - p = 0 , 0024 , 3h - p = 0 , 003 ; CD curve : 2h and 3h p < 0 , 0001 ( B ) UC curve : 4h - p = 0 , 0186 , 5h - p = 0 , 0393 , 6h - p = 0 , 039 , 8h - p < 0 , 0001 ; CD curve : 4h - p = 0 , 0001 , 5h - p = 0 , 0028 , 6h - p = 0 , 0024 , 8h - p < 0 , 0001 . ( C ) 3h - p = 0 , 0422 , 4h - p = 0 , 0422 , 6h - p = 0 , 0012 , 8h - p < 0 , 0001 . ( F - I ) Optical density ( 600nm ) during in vitro growth assay of Escherichia coli ( 25922 strain from ATCC ) co - cultured with stool - purified IgA1 ( F ) or IgA2 ( G ) from non - IBD ( n = 2 ) and CD ( n = 2 ) . All patient samples ( biological replicates ) have been tested in technical duplicates . Comparison between IgA1 and IgA2 co - culture in non - IBD ( H ) , and CD ( I ) . These IgA samples were from the same patients throughout each experiment . Two - way ANOVA with Holm - Sidak correction after D ' Agostino - Pearson nomality test , p - values are as follows : ( F ) Grey curve : 4h - p = 0 , 0004 , 5h - p = 0 , 0014 ; Black curve : 4h and 5h - p < 0 , 0001 . ( G ) De - glycosylated CD vs . De - glycosylated non - IBD : 4h - p = 0 , 0003 , 5h - p < 0 , 0001 . ( H ) Native vs . de - glycosylated : 4h and 5h - p < 0 , 0001 ; De - glycosylated IgA1 vs . De - glycosylated IgA2 : 5h - p = 0 , 0012 . ( H ) Native vs . de - glycosylated : 4h - p = 0 , 001 and 5h - p = 0 , 006 ; De - glycosylated IgA1 vs . De - glycosylated IgA2 : 5h - p < 0 , 0001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A-E) Optical density (600nm) variations during in vitro growth assay of Salmonella enterica Typhimurium (SL1344) co-cultured with stool-purified IgA1 (A) or IgA2 (B) from non-IBD (n=4), CD (n=6) and UC (n=3). Comparison between IgA1 and IgA2 co-culture in non-IBD (C), CD (D), and UC (E). Two-way ANOVA with Holm-Sidak correction after D'Agostino-Pearson nomality test, p-values are as follows: (A) UC curve: 2h - p=0,0024, 3h - p=0,003; CD curve: 2h and 3h p<0,0001 (B) UC curve: 4h - p=0,0186, 5h - p=0,0393, 6h - p=0,039, 8h - p<0,0001; CD curve: 4h - p=0,0001, 5h - p=0,0028, 6h - p=0,0024, 8h - p<0,0001. (C) 3h - p=0,0422, 4h - p=0,0422, 6h - p=0,0012, 8h - p<0,0001.(F-I) Optical density (600nm) during in vitro growth assay of Escherichia coli (25922 strain from ATCC) co-cultured with stool-purified IgA1 (F) or IgA2 (G) from non-IBD (n=2) and CD (n=2). All patient samples (biological replicates) have been tested in technical duplicates. Comparison between IgA1 and IgA2 co-culture in non-IBD (H), and CD (I). These IgA samples were from the same patients throughout each experiment. Two-way ANOVA with Holm-Sidak correction after D'Agostino-Pearson nomality test, p-values are as follows: (F) Grey curve: 4h - p=0,0004, 5h - p=0,0014; Black curve: 4h and 5h - p<0,0001. (G) De-glycosylated CD vs. De-glycosylated non-IBD: 4h - p=0,0003, 5h - p<0,0001. (H) Native vs. de-glycosylated: 4h and 5h - p<0,0001; De-glycosylated IgA1 vs. De-glycosylated IgA2: 5h - p=0,0012. (H) Native vs. de-glycosylated: 4h - p=0,001 and 5h - p= 0,006; De-glycosylated IgA1 vs. De-glycosylated IgA2: 5h - p<0,0001.


Figure 6 ( A ) Combined FISH - IF staining for each probe . ( B ) Total fluorescence detected per probe and per biopsy . Erec482 - * : p = 0 , 00233 ; Ato291 - * : p = 0 , 0268 ; Bac303 - * : p = 0 , 0196 . ( C ) Percent of co - occurred IgA2 and FISH - bound particles . Erec482 - * : p = 0 , 0073 . Two - way ANOVA with Holm - Sidak correction after D ' Agostino - Pearson nomality test . Non - IBD : n = 3 ; Active CD : n = 6 ; Inactive CD : n = 4 . All patient samples ( biological replicates ) have been tested in technical duplicates .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Combined FISH-IF staining for each probe. (B) Total fluorescence detected per probe and per biopsy. Erec482 - *: p=0,00233; Ato291 - *: p=0,0268; Bac303 - *: p=0,0196. (C) Percent of co-occurred IgA2 and FISH-bound particles. Erec482 - *: p=0, 0073.Two-way ANOVA with Holm-Sidak correction after D'Agostino-Pearson nomality test. Non-IBD: n = 3; Active CD: n= 6; Inactive CD: n=4. All patient samples (biological replicates) have been tested in technical duplicates.


Figure 1A . MS - proteomic analysis of RCS chondrocytes treated with vehicle ( 5 % ABS ) and FGF18 ( 50 ng / mL ) for 16 hours . Biological processes and cellular components regulated by FGF signaling are shown . FDR < 0 . 05 . N = 4 biological replicates / treatment were analyzed . p - value was calculated using 1D annotation enrichment test based on Wilcoxon - Mann - Whitney test . FACS analysis of Lysotracker ( B ) dye fluorescence in RCS treated with the indicated FGF ligands ( 50 ng / mL ) for 16h . Fluorescence intensities were expressed as % relative to vehicle ( 5 % ABS ) . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates / treatment . Analysis of variance one way ( ANOVA ) p < 0 . 002 ( B ) Tukey ' s post hoc test * * * p < 0 . 0005 . FACS analysis of DQ - BSA ( C ) dye fluorescence in RCS treated with the indicated FGF ligands ( 50 ng / mL ) for 16h . Concanamycin A was used at 100 nM for 1h to inhibit lysosomal function . Fluorescence intensities were expressed as % relative to vehicle ( 5 % ABS ) . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates / treatment . Analysis of variance one way ( ANOVA ) = 0 . 005 ( C ) ; Tukey ' s post hoc test * * * p < 0 . 0005 . D . Lamp1 immunofluorescence ( red ) in RCS chondrocytes treated with vehicle ( 5 % ABS ) and FGF18 ( 50 ng / mL ) for 16 hours . Insets show magnification of the boxed area . Nuclei were stained with DAPI ( blue ) . Scale bar 15 μm and 2 μm ( higher magnification boxes ) . Bar graph shows quantification of Lamp1 - positive vesicles / cell . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates / treatment . n = 45 cells were analyzed . Student paired T - Test * p < 0 . 05 . E . Representative TEM images of RCS chondrocytes treated with 5 % ABS ( vehicle ) and FGF18 ( 50 ng / mL ) for 16 hours . BafA1 ( 100 nM ; 4 hours ) was used to inhibit lysosome activity . Arrows indicate lysosomes . Higher magnification insets showed presence of ER membranes decorated with ribosomes ( arrowheads ) . Scale bar 500nm . Quantification shows average number of ER membranes and mitochondria number / lysosome vesicle ( Lys ) . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates / treatment . ER fragment / vesicle n = 60 ( vehicle ) and n = 72 ( FGF18 ) cells were analyzed ; Mitochondria number / vesicle : n = 40 ( vehicle ) and n = 72 ( FGF18 ) cells were analyzed . Student paired T - Test * * * p < 0 . 0005 ; NS not significant . F . Representative co - immunofluorescence staining of ER ( ER tracker BODIPY green ) and lysosomes ( Lamp1 , red ) in RCS chondrocytes treated with vehicle ( 5 % ABS ) and FGF18 ( 50 ng / mL ) for 16 hours . Insets show higher magnification of boxed area . Scale bar 15 μm and 2 μm ( higher magnification boxes ) . N = 3 biological replicates / treatment . G . Schematic representation of EATR assay : eGFP fluorescence , but not mCherry , is lost at acidic pH . Chondrocytes with indicated genotypes ( ctrl = wild type ) were treated with FGF18 ( 50 ng / mL ; 12 hours ) and BafA1 ( 200 nM ; 3 hours ) where indicated . ER acidification was measured by FACS . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates . Analysis of variance one way ( ANOVA ) p < 0 . 0001 ; Tukey ' s post hoc test * * * p < 0 . 0005 ; * * p < 0 . 005 ; NS not significant . H . FACS analysis of DQ - BSA dye fluorescence in chondrocytes with indicated genotypes ( ctrl = wild type ) treated with FGF18 ( 50 ng / mL ) for 16 hours . Concanamycin A was used at 100 nM for 1h to inhibit lysosomal degradation . Fluorescence intensities were expressed as % relative to vehicle ( 5 % ABS ) . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates / treatment / genotype . Analysis of variance ( ANOVA ) p = 0 . 00279 ; Tukey ' s post hoc test * * p < 0 . 005 ; * p < 0 . 05 ; NS not significant . I . EATR assay . Chondrocytes with indicated genotypes ( ctrl = wild type ) treated with FGF18 ( 50 ng / mL ; 16 hours ) where indicated . ER acidification was measured by FACS . Mean + / - standard error of the mean ( sem ) of N = 17 ( veh ) , N = 17 ( FGF18 ) , N = 4 ( FGFR3 ; 4KO ) ; N = 4 ( FGFR3 ; 4KO FGF18 ) biological replicates . Analysis of variance one way ( ANOVA ) p < 0 . 0001 ; Tukey ' s post hoc test * * * p < 0 . 0005 ; NS not significant . J . Representative immunofluorescence staining of ER ( CLIMP63 , green ) and lysosomes ( TMEM192 - HA , red ) in chondrocytes with indicated genotypes ( ctrl = wild type ) treated with FGF18 ( 50 ng / mL for 16h ) . BafA1 was used at 100 nM for 4 hours to inhibit lysosomal degradation . Quantification of relative CLIMP63 fluorescence into TMEM192 - HA positive vesicles . N = 3 biological replicates ; n = 21 cells were analyzed . Mean + / - standard error of the mean ( sem ) . Paired Student ' s T - test NS = not significant . Scale bar 10 μm and 1 . 3 μm ( higher magnification boxes ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. MS-proteomic analysis of RCS chondrocytes treated with vehicle (5% ABS) and FGF18 (50 ng/mL) for 16 hours. Biological processes and cellular components regulated by FGF signaling are shown. FDR <0.05. N=4 biological replicates/treatment were analyzed. p-value was calculated using 1D annotation enrichment test based on Wilcoxon-Mann-Whitney test.FACS analysis of Lysotracker (B) dye fluorescence in RCS treated with the indicated FGF ligands (50 ng/mL) for 16h. Fluorescence intensities were expressed as % relative to vehicle (5% ABS). Mean +/- standard error of the mean (sem) of N=3 biological replicates/treatment. Analysis of variance one way (ANOVA) p<0.002 (B) Tukey's post hoc test ***p<0.0005.FACS analysis of DQ-BSA (C) dye fluorescence in RCS treated with the indicated FGF ligands (50 ng/mL) for 16h. Concanamycin A was used at 100 nM for 1h to inhibit lysosomal function. Fluorescence intensities were expressed as % relative to vehicle (5% ABS). Mean +/- standard error of the mean (sem) of N=3 biological replicates/treatment. Analysis of variance one way (ANOVA) =0.005 (C); Tukey's post hoc test ***p<0.0005.D. Lamp1 immunofluorescence (red) in RCS chondrocytes treated with vehicle (5% ABS) and FGF18 (50 ng/mL) for 16 hours. Insets show magnification of the boxed area. Nuclei were stained with DAPI (blue). Scale bar 15 μm and 2 μm (higher magnification boxes) . Bar graph shows quantification of Lamp1-positive vesicles/cell. Mean +/- standard error of the mean (sem) of N=3 biological replicates/treatment. n=45 cells were analyzed. Student paired T-Test *p<0.05.E. Representative TEM images of RCS chondrocytes treated with 5% ABS (vehicle) and FGF18 (50 ng/mL) for 16 hours. BafA1 (100 nM; 4 hours) was used to inhibit lysosome activity. Arrows indicate lysosomes. Higher magnification insets showed presence of ER membranes decorated with ribosomes (arrowheads). Scale bar 500nm. Quantification shows average number of ER membranes and mitochondria number/ lysosome vesicle (Lys). Mean +/- standard error of the mean (sem) of N=3 biological replicates/treatment. ER fragment/vesicle n=60 (vehicle) and n=72 (FGF18) cells were analyzed; Mitochondria number/vesicle: n=40 (vehicle) and n=72 (FGF18) cells were analyzed. Student paired T-Test ***p<0.0005; NS not significant.F. Representative co-immunofluorescence staining of ER (ER tracker BODIPY green) and lysosomes (Lamp1, red) in RCS chondrocytes treated with vehicle (5% ABS) and FGF18 (50 ng/mL) for 16 hours. Insets show higher magnification of boxed area. Scale bar 15 μm and 2 μm (higher magnification boxes). N=3 biological replicates/treatment.G. Schematic representation of EATR assay: eGFP fluorescence, but not mCherry, is lost at acidic pH. Chondrocytes with indicated genotypes (ctrl=wild type) were treated with FGF18 (50 ng/mL; 12 hours) and BafA1 (200 nM; 3 hours) where indicated. ER acidification was measured by FACS. Mean +/- standard error of the mean (sem) of N=3 biological replicates. Analysis of variance one way (ANOVA) p<0.0001; Tukey's post hoc test ***p<0.0005; **p<0.005; NS not significant.H. FACS analysis of DQ-BSA dye fluorescence in chondrocytes with indicated genotypes (ctrl=wild type) treated with FGF18 (50 ng/mL) for 16 hours. Concanamycin A was used at 100 nM for 1h to inhibit lysosomal degradation. Fluorescence intensities were expressed as % relative to vehicle (5% ABS). Mean +/- standard error of the mean (sem) of N=3 biological replicates/treatment/genotype. Analysis of variance (ANOVA) p=0.00279; Tukey's post hoc test **p<0.005; *p<0.05; NS not significant.I. EATR assay. Chondrocytes with indicated genotypes (ctrl=wild type) treated with FGF18 (50 ng/mL; 16 hours) where indicated. ER acidification was measured by FACS. Mean +/- standard error of the mean (sem) of N= 17 (veh), N=17 (FGF18), N=4 (FGFR3;4KO); N=4 (FGFR3;4KO FGF18) biological replicates. Analysis of variance one way (ANOVA) p<0.0001; Tukey's post hoc test ***p<0.0005; NS not significant.J. Representative immunofluorescence staining of ER (CLIMP63, green) and lysosomes (TMEM192-HA, red) in chondrocytes with indicated genotypes (ctrl=wild type) treated with FGF18 (50 ng/mL for 16h). BafA1 was used at 100 nM for 4 hours to inhibit lysosomal degradation. Quantification of relative CLIMP63 fluorescence into TMEM192-HA positive vesicles. N=3 biological replicates; n=21 cells were analyzed. Mean +/- standard error of the mean (sem). Paired Student's T-test NS= not significant. Scale bar 10 μm and 1.3 μm (higher magnification boxes).


Figure 2A . MS - phospho - proteomics analysis of RCS with indicated genotypes ( ctrl = wild type ) treated with vehicle ( 5 % ABS ) and FGF18 ( 50 ng / mL ) for 16 hours , showing biological processes regulated by FGF signaling ( in blue : up - regulated , in green : down - regulated ) . N = 4 biological replicates were analyzed . FDR < 0 . 05 . Representative western blot analysis of IRS1 , p - P70S6K ( T389 ) , P70S6K , p - AKT ( T308 ) , AKT in RCS chondrocytes treated with vehicle ( 5 % ABS ) and FGF18 ( 50 ng / mL ) for 12h . MG132 ( 10 μM for 6h ) to inhibit proteasome ( C ) activity , respectively . β - actin was used as loading control . N = 3 independent experiments . Bar graphs showed quantification of indicated proteins normalized to their totals . IRS1 was normalized to β - actin . Mean + / - standard error of the mean ( sem ) . Student ' s paired T - test * * p < 0 . 005 ; * p < 0 . 05 ; NS not significant . Representative western blot analysis of IRS1 , p - P70S6K ( T389 ) , P70S6K , p - AKT ( T308 ) , AKT , p - cJUN ( s73 ) and cJUN in RCS chondrocytes treated with vehicle ( 5 % ABS ) and FGF18 ( 50 ng / mL ) for 12h . JNK inhibitor ( 50 μM for 12h ) to inhibit JNK ( D ) activity , respectively . β - actin was used as loading control . N = 3 independent experiments . Bar graphs showed quantification of indicated proteins normalized to their totals . IRS1 was normalized to β - actin . Mean + / - standard error of the mean ( sem ) . Student ' s paired T - test * * p < 0 . 005 ; * p < 0 . 05 ; NS not significant . E . Co - immunofluorescence of p - IRS1 S307 mouse ( green ) and p - S6 S240 / S242 ( red ) ribosomal protein in IRS1 - overexpressing RCS chondrocytes treated with FGF18 ( 50 ng / mL ) for 12h . Nuclei ( N ) were stained with DAPI ( blue ) . Scale bar 15 μm . Quantification analysis of p - S6 fluorescence intensity in IRS1 - overexpressing vs non expressing RCS chondrocytes . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates . n = 35 cells analyzed . Student ' s paired T - test * p < 0 . 05 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. MS-phospho-proteomics analysis of RCS with indicated genotypes (ctrl=wild type) treated with vehicle (5% ABS) and FGF18 (50 ng/mL) for 16 hours, showing biological processes regulated by FGF signaling (in blue: up-regulated, in green: down-regulated). N=4 biological replicates were analyzed. FDR <0.05.Representative western blot analysis of IRS1, p-P70S6K (T389), P70S6K, p-AKT (T308), AKT in RCS chondrocytes treated with vehicle (5% ABS) and FGF18 (50 ng/mL) for 12h. MG132 (10 μM for 6h) to inhibit proteasome (C) activity, respectively. β-actin was used as loading control. N=3 independent experiments. Bar graphs showed quantification of indicated proteins normalized to their totals. IRS1 was normalized to β-actin. Mean +/- standard error of the mean (sem). Student's paired T-test **p<0.005; *p<0.05; NS not significant.Representative western blot analysis of IRS1, p-P70S6K (T389), P70S6K, p-AKT (T308), AKT, p-cJUN (s73) and cJUN in RCS chondrocytes treated with vehicle (5% ABS) and FGF18 (50 ng/mL) for 12h. JNK inhibitor (50 μM for 12h) to inhibit JNK (D) activity, respectively. β-actin was used as loading control. N=3 independent experiments. Bar graphs showed quantification of indicated proteins normalized to their totals. IRS1 was normalized to β-actin. Mean +/- standard error of the mean (sem). Student's paired T-test **p<0.005; *p<0.05; NS not significant.E. Co-immunofluorescence of p-IRS1 S307 mouse (green) and p-S6 S240/S242 (red) ribosomal protein in IRS1-overexpressing RCS chondrocytes treated with FGF18 (50 ng/mL) for 12h. Nuclei (N) were stained with DAPI (blue). Scale bar 15 μm. Quantification analysis of p-S6 fluorescence intensity in IRS1-overexpressing vs non expressing RCS chondrocytes. Mean +/- standard error of the mean (sem) of N=3 biological replicates. n=35 cells analyzed. Student's paired T-test *p<0.05.


Figure 3A . EATR assay . Chondrocytes were treated with FGF18 ( 50 ng / mL ; 16 hours ) and Actinomycin ( 1μg / mL ; last 4 hours ) where indicated . ER acidification was measured by FACS . Mean + / - standard error of the mean ( sem ) of N = 17 ( veh ) , N = 17 ( FGF18 ) , N = 5 ( actinomycin ) , N = 5 ( actinomycin + FGF18 ) biological replicates . Analysis of variance one way ( ANOVA ) p < 0 . 0001 ; Tukey ' s post hoc test * * * p < 0 . 0005 ; NS not significant . B . FACS analysis of Lysotracker dye fluorescence in RCS chondrocytes treated with FGF18 ( 50 ng / mL ) for 16 hours . Actinomycin ( 1μg / mL ) was added for the last 4 hours . Fluorescence intensities were calculated as % relative to vehicle ( 5 % ABS ) . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates . Analysis of variance one way ( ANOVA ) p = 0 . 0002 ; Tukey ' s post hoc test * * * p < 0 . 0005 ; NS not significant . C . Representative immunofluorescence staining of Lamp1 ( red ) in RCS chondrocytes treated with vehicle ( 5 % ABS ) , FGF18 ( 50 ng / mL ; 16 hours ) and Actinomycin ( 1μg / mL ; 4 hours ) . Nuclei were stained with DAPI ( blue ) . Scale bar 10 μm . Representative images of N = 3 biological replicates . Scale bar 10 μm . D . Heat - map of lysosomal and autophagy genes expression in RCS chondrocytes with indicated genotypes ( ctrl = wild type ) , treated with vehicle ( 5 % ABS ) and FGF18 ( 50 ng / mL ) for 16 hours . FDR < 0 . 05 . N = 3 biological replicates / treatment were analyzed . In green : down - regulated , in red : up - regulated gene expression . E . qRT - PCR analysis of ER - phagy receptors in RCS chondrocytes . Gene expression was analyzed after FGF18 ( 50 ng / mL ) treatment for 16 hours . Fold change values were relative to vehicle ( 5 % ABS ) and normalized to Cyclophilin gene . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates / treatment . Student ' s paired T - test * * p < 0 . 005 ; * p < 0 . 05 . F . ( top ) Representative model of Fam134b ∆ LIR protein . LIR = LC3 - Interacting - Region . ( bottom ) Western blot analysis of Fam134b in chondrocytes with indicated genotypes ( ctrl = wild type ) treated with FGF18 ( 50 ng / mL ) for 16 hours . Representative images of N = 3 biological replicates / treatment . β - actin was used as loading control . Bar graph showed quantification of Fam134b band intensity normalized to β - actin . Mean + / - standard error of the mean ( sem ) . Student ' s paired T - test , * p < 0 . 05 . G . Immunofluorescence staining of CLIMP63 ( green ) and lysosomes ( TMEM192 - HA , red ) in RCS chondrocytes with indicated genotypes ( ctrl = wild type ) upon FGF18 treatment ( 50 ng / mL for 16h ) . BafA1 ( 100 nM ; 4 hours ) was used to inhibit lysosomal degradation ; scale bar 20 μm . Insets show magnification of CLIMP63 accumulation into lysosomes ; scale bar 2 μm . Quantification of CLIMP63 fluorescence intensity into TMEM192 - HA decorated lysosomes . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates . n = 10 cells / experiment were analyzed . Analysis of variance one way ( ANOVA ) p = 1 . 55e - 7 ; Tukey ' s post hoc test * * * p < 0 . 0005 ; NS not significant . H . EATR assay in RCS with indicated genotypes ( ctrl = wild type ) showing % of cells with acidified ER by FACS analysis . FGF18 was used at 50 ng / mL for 16 hours . Mean + / - standard error of the mean ( sem ) of N = 4 biological replicates / treatment / genotype . Analysis of variance one way ( ANOVA ) p < 0 . 0001 ; Tukey ' s post hoc test * * * p < 0 . 0005 ; NS not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. EATR assay. Chondrocytes were treated with FGF18 (50 ng/mL; 16 hours) and Actinomycin (1μg/mL; last 4 hours) where indicated. ER acidification was measured by FACS. Mean +/- standard error of the mean (sem) of N=17 (veh), N=17 (FGF18), N=5 (actinomycin), N=5 (actinomycin + FGF18) biological replicates. Analysis of variance one way (ANOVA) p<0.0001; Tukey's post hoc test ***p<0.0005; NS not significant.B. FACS analysis of Lysotracker dye fluorescence in RCS chondrocytes treated with FGF18 (50 ng/mL) for 16 hours. Actinomycin (1μg/mL) was added for the last 4 hours. Fluorescence intensities were calculated as % relative to vehicle (5% ABS). Mean +/- standard error of the mean (sem) of N=3 biological replicates. Analysis of variance one way (ANOVA) p=0.0002; Tukey's post hoc test ***p<0.0005; NS not significant.C. Representative immunofluorescence staining of Lamp1 (red) in RCS chondrocytes treated with vehicle (5% ABS), FGF18 (50 ng/mL; 16 hours) and Actinomycin (1μg/mL; 4 hours). Nuclei were stained with DAPI (blue). Scale bar 10 μm. Representative images of N=3 biological replicates. Scale bar 10 μm.D. Heat-map of lysosomal and autophagy genes expression in RCS chondrocytes with indicated genotypes (ctrl=wild type), treated with vehicle (5% ABS) and FGF18 (50 ng/mL) for 16 hours. FDR <0.05. N=3 biological replicates/treatment were analyzed. In green: down-regulated, in red: up-regulated gene expression.E. qRT-PCR analysis of ER-phagy receptors in RCS chondrocytes. Gene expression was analyzed after FGF18 (50 ng/mL) treatment for 16 hours. Fold change values were relative to vehicle (5% ABS) and normalized to Cyclophilin gene. Mean +/- standard error of the mean (sem) of N=3 biological replicates/treatment. Student's paired T-test **p<0.005; *p<0.05.F. (top) Representative model of Fam134b∆LIR protein. LIR= LC3-Interacting-Region. (bottom) Western blot analysis of Fam134b in chondrocytes with indicated genotypes (ctrl=wild type) treated with FGF18 (50 ng/mL) for 16 hours. Representative images of N=3 biological replicates/treatment. β-actin was used as loading control. Bar graph showed quantification of Fam134b band intensity normalized to β-actin. Mean +/- standard error of the mean (sem). Student's paired T-test, *p<0.05.G. Immunofluorescence staining of CLIMP63 (green) and lysosomes (TMEM192-HA, red) in RCS chondrocytes with indicated genotypes (ctrl=wild type) upon FGF18 treatment (50 ng/mL for 16h). BafA1 (100 nM; 4 hours) was used to inhibit lysosomal degradation; scale bar 20 μm. Insets show magnification of CLIMP63 accumulation into lysosomes; scale bar 2 μm. Quantification of CLIMP63 fluorescence intensity into TMEM192-HA decorated lysosomes. Mean +/- standard error of the mean (sem) of N=3 biological replicates. n=10 cells/experiment were analyzed. Analysis of variance one way (ANOVA) p=1.55e-7; Tukey's post hoc test ***p<0.0005; NS not significant. H. EATR assay in RCS with indicated genotypes (ctrl=wild type) showing % of cells with acidified ER by FACS analysis. FGF18 was used at 50 ng/mL for 16 hours. Mean +/- standard error of the mean (sem) of N=4 biological replicates/treatment/genotype. Analysis of variance one way (ANOVA) p<0.0001; Tukey's post hoc test ***p<0.0005; NS not significant.


Figure 4A . TFE3 ( green ) subcellular localization in RCS with indicated genotypes ( control = wild type ) treated with FGF18 ( 50 ng / mL ) for 16 hours . Torin1 ( 1 μM for 2 hours ) was used as positive control . Nuclei were stained with DAPI ( blue ) . Bar graph shows quantification ( expressed as % ) of cells with nuclear TFE3 and TFEB ( representative images of TFEB immunofluorescence are shown in EV2E ) . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates ; n = 80 ( TFE3 ) and n = 70 ( TFEB ) cells / experiment were analyzed . Scale bar 10 μm . Analysis of variance one - way ( ANOVA ) p = 3 . 23e - 12 ( TFEB ) , p = 2 . 48e - 11 ( TFE3 ) ; Tukey ' s post hoc test * * * p < 0 . 0005 ; NS not significant . B . Western blot analysis of TFEB , and phospo - TFEB ( Serine 142 ) in RCS chondrocytes stably expressing human TFEB - 3XFlag protein . Cells were treated with vehicle ( 5 % ABS ) or FGF18 ( 50 ng / mL ) for 16h . β - actin was used as loading control . Representative of three independent experiments . C . GFP immuno - EM staining in FGFR3 / 4KO chondrocytes infected with a constitutive nuclear ( and active ) mutant TFEB fused to GFP tag ( TFEB - S142A : S211A - GFP ) . GFP positive gold immune - particles showed presence of GFP - puncta into the nucleus ( N , stained in green for visualization ) . Lysosomes were stained in blue for visualization . Insets showed magnification of lysosomes . Quantification of lysosome diameter ( nm ) in control ( wild type ) and FGFR3 ; 4KO chondrocytes infected with empty or with TFEB - S142A : S211A - GFP vector . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates . n = 40 ( veh ) n = 78 ( FGFR3 ; 4KO ) n = 57 ( FGFR3 ; 4KO + TFEB - S142A : S211A - GFP ) cells were analyzed . Scale bar 500 nm . Kruskal Wallis test p = 1 . 43e - 13 ; Nemenyi post hoc test * * * p < 0 . 0005 ; NS not significant . D . Co - immunofluorescence of p - IRS1 ( S307 mouse ) and TFEB in IRS1 - overexpressing RCS chondrocytes treated with FGF18 ( 50 ng / mL ) for 12h . Nuclei ( N ) were stained with DAPI ( blue ) and delimited with dashed line . Scale bar 15 μm . Quantification of TFEB nuclear localization in IRS1 - overexpressing vs non - expressing RCS . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates . n = 124 cells analyzed ; Student ' s un - paired T - test * p < 0 . 05 . E . Subcellular localization analysis of TFEB ( red ) and TFE3 ( green ) in RCS chondrocytes treated with FGF18 ( 50 ng / mL ) for 12h . JNK inhibitor was used at 50 μM for 12h . Nuclei were stained with DAPI ( blue ) . Quantification analysis showed % of cells with nuclear TFEB and TFE3 in RCS chondrocytes with indicated treatments . Scale bar 15 μm . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates . n = 126 cells ( control ) , 126 cells ( FGF18 ) , 95 cells ( JNK Inhibitor ) , 163 cells ( JNK Inhibitor + FGF18 ) . Student ' s paired T - test * p < 0 . 05 . F . Western blot analysis of phospho - TFEB ( S142 ) and IRS1 in RCS chondrocytes treated with vehicle ( 5 % ABS ) and FGF18 ( 50 ng / mL ) for 12h . JNK inhibitor was used at 50 μM for 12h to inhibit kinase activity . β - actin was used as loading control . Representative images of N = 3 independent experiments . Bar graphs show quantification of indicated proteins normalized to β - actin . Mean + / - standard error of the mean ( sem ) . Student ' s paired T - test * p < 0 . 05 ; NS not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. TFE3 (green) subcellular localization in RCS with indicated genotypes (control=wild type) treated with FGF18 (50 ng/mL) for 16 hours. Torin1 (1 μM for 2 hours) was used as positive control. Nuclei were stained with DAPI (blue). Bar graph shows quantification (expressed as %) of cells with nuclear TFE3 and TFEB (representative images of TFEB immunofluorescence are shown in EV2E). Mean +/- standard error of the mean (sem) of N=3 biological replicates; n=80 (TFE3) and n=70 (TFEB) cells/experiment were analyzed. Scale bar 10 μm. Analysis of variance one-way (ANOVA) p=3.23e-12 (TFEB), p=2.48e-11 (TFE3); Tukey's post hoc test ***p<0.0005; NS not significant.B. Western blot analysis of TFEB, and phospo-TFEB (Serine 142) in RCS chondrocytes stably expressing human TFEB-3XFlag protein. Cells were treated with vehicle (5% ABS) or FGF18 (50 ng/mL) for 16h. β-actin was used as loading control. Representative of three independent experiments.C. GFP immuno-EM staining in FGFR3/4KO chondrocytes infected with a constitutive nuclear (and active) mutant TFEB fused to GFP tag (TFEB- S142A:S211A-GFP). GFP positive gold immune-particles showed presence of GFP-puncta into the nucleus (N, stained in green for visualization). Lysosomes were stained in blue for visualization. Insets showed magnification of lysosomes. Quantification of lysosome diameter (nm) in control (wild type) and FGFR3;4KO chondrocytes infected with empty or with TFEB- S142A:S211A-GFP vector. Mean +/- standard error of the mean (sem) of N=3 biological replicates. n=40 (veh) n=78 (FGFR3;4KO) n=57 (FGFR3;4KO +TFEB-S142A:S211A-GFP) cells were analyzed. Scale bar 500 nm. Kruskal Wallis test p=1.43e-13; Nemenyi post hoc test ***p<0.0005; NS not significant.D. Co-immunofluorescence of p-IRS1 (S307 mouse) and TFEB in IRS1-overexpressing RCS chondrocytes treated with FGF18 (50 ng/mL) for 12h. Nuclei (N) were stained with DAPI (blue) and delimited with dashed line. Scale bar 15 μm. Quantification of TFEB nuclear localization in IRS1-overexpressing vs non-expressing RCS. Mean +/- standard error of the mean (sem) of N=3 biological replicates. n= 124 cells analyzed; Student's un-paired T-test *p<0.05.E. Subcellular localization analysis of TFEB (red) and TFE3 (green) in RCS chondrocytes treated with FGF18 (50 ng/mL) for 12h. JNK inhibitor was used at 50 μM for 12h. Nuclei were stained with DAPI (blue). Quantification analysis showed % of cells with nuclear TFEB and TFE3 in RCS chondrocytes with indicated treatments. Scale bar 15 μm. Mean +/- standard error of the mean (sem) of N=3 biological replicates. n= 126 cells (control), 126 cells (FGF18), 95 cells (JNK Inhibitor), 163 cells (JNK Inhibitor + FGF18). Student's paired T-test *p<0.05.F. Western blot analysis of phospho-TFEB (S142) and IRS1 in RCS chondrocytes treated with vehicle (5% ABS) and FGF18 (50 ng/mL) for 12h. JNK inhibitor was used at 50 μM for 12h to inhibit kinase activity. β-actin was used as loading control. Representative images of N=3 independent experiments. Bar graphs show quantification of indicated proteins normalized to β-actin. Mean +/- standard error of the mean (sem). Student's paired T-test *p<0.05; NS not significant.


Figure 5A . qRT - PCR analysis of Fam134b gene expression in chondrocytes with indicated genotypes ( ctrl = wild type ) treated with vehicle ( 5 % ABS ) or with FGF18 ( 50 ng / mL ; 16 hours ) . Fold change values were relative to vehicle and normalized to Cyclophilin gene . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates . Analysis of variance one way ( ANOVA ) p < 0 . 0001 : Tukey ' s post hoc test * * * p < 0 . 0005 ; NS not significant . B . Western blot analysis of Fam134b protein in chondrocytes with indicated genotypes treated with vehicle ( 5 % ABS ) and FGF18 ( 50 ng / mL ) overnight . β actin was used as loading control . Representative image of N = 3 biological replicates . Bar graph showed quantification of Fam134b normalized to β - actin . Analysis of variance one way ( ANOVA ) p < 0 . 0001 ; Sidak ' s multiple comparison test * * * p < 0 . 0005 ; * * p < 0 . 005 NS not significant . C . Co - staining of CLIMP63 ( green ) and TMEM192 - HA ( lysosomes , red ) in control and TFEB ; 3KO RCS treated with FGF18 ( 50 ng / mL for 16 hours ) . BaFA1 was used at 100 nM for 3h . Scale bar 15 μm and 2 μm ( higher magnification boxes ) . D . Quantification of CLIMP63 fluorescence intensity into TMEM192 - HA decorated lysosomes . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates / treatment / genotype ( ctrl = wild type ) . n = 10 cells / experiment were analyzed . Analysis of variance one way ( ANOVA ) p = 1 . 55e - 7 ; Tukey ' s post hoc test * * * p < 0 . 0005 ; NS not significant . E . EATR assay in chondrocytes with indicated genotypes ( ctrl = wild type ) showing % of cells with acidified ER measured by FACS . FGF18 was used at 50 ng / mL overnight . Mean + / - standard error of the mean ( sem ) of N = 4 biological replicates . Analysis of variance one way ( ANOVA ) p < 0 . 0001 : Tukey ' s post hoc test * * * p < 0 . 0005 ; * p < 0 . 05 ; NS not significant . F , G Data plots show quantification of mCherry + vesicles / cell ( autolysosomes ) ( F ) and mCherry + / GFP + vesicles / cell ( autophagosomes ) ( G ) in wild type ( ctrl ) and TFEB ; 3KO cells treated with vehicle ( veh ) or FGF18 . N = 3 independent experiments . Mean + / - standard error of the mean ( sem ) of N = 24 ( wild type treated with 5 % ABS , veh ) , N = 30 ( wild type treated with FGF18 ) , N = 27 ( TFEB ; 3KO veh ) , N = 33 ( TFEB ; 3KO FGF18 ) cells . Student un - paired T - Test * * * p < 0 . 0005 ; * * p < 0 . 005 . H . ChIP analysis of TFEB binding to Fam134b DNA in RCS cells transfected with TFEB - 3XFLAG . Numbers in the CLEAR site ( yellow box ) refer to the distance [ in base pairs ] from the transcriptional start site ( + 1 ) of Fam134b - 2 gene . Immunoprecipitated DNA was normalized to the input and plotted as relative enrichment over a mock control . Bar graph shows fold change enrichment ; mean + / - standard error of the mean ( sem ) of N = 3 independent experiments . Student un - paired T - Test * * p < 0 . 005 . I - J . Luciferase assays in RCS chondrocytes using as promoter a 0 , 7kb genomic Fam134b DNA fragment containing a wild type ( FAM134B - WT ) or a deleted ( FAM134B - mut ) version of the CLEAR site . TFEB plasmid transfection amount and FGF18 ( 50 ng / mL for 16h ) treatments are indicated . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates . Student paired T - test * p < 0 . 05 ; * * p < 0 . 005 . K . Luciferase activity in wild type ( ctrl ) and TFEB ; 3KO RCS chondrocytes with indicated genotypes expressing the indicated Fam134b luciferase report plasmids and treated with FGF18 overnight ( 50 ng / mL ) where indicated . Mean + / - standard error of the mean ( sem ) of N = 5 biological replicates . Analysis of variance one way ( ANOVA ) p < 0 . 0001 : Sidak ' s multiple comparison test * * * p < 0 . 0005 ; NS not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. qRT-PCR analysis of Fam134b gene expression in chondrocytes with indicated genotypes (ctrl=wild type) treated with vehicle (5% ABS) or with FGF18 (50 ng/mL; 16 hours). Fold change values were relative to vehicle and normalized to Cyclophilin gene. Mean +/- standard error of the mean (sem) of N=3 biological replicates. Analysis of variance one way (ANOVA) p<0.0001: Tukey's post hoc test ***p<0.0005; NS not significant.B. Western blot analysis of Fam134b protein in chondrocytes with indicated genotypes treated with vehicle (5% ABS) and FGF18 (50 ng/mL) overnight. β actin was used as loading control. Representative image of N=3 biological replicates. Bar graph showed quantification of Fam134b normalized to β-actin. Analysis of variance one way (ANOVA) p<0.0001; Sidak's multiple comparison test ***p<0.0005; **p<0.005 NS not significant.C. Co-staining of CLIMP63 (green) and TMEM192-HA (lysosomes, red) in control and TFEB;3KO RCS treated with FGF18 (50 ng/mL for 16 hours). BaFA1 was used at 100 nM for 3h. Scale bar 15 μm and 2 μm (higher magnification boxes).D. Quantification of CLIMP63 fluorescence intensity into TMEM192-HA decorated lysosomes. Mean +/- standard error of the mean (sem) of N=3 biological replicates/treatment/genotype (ctrl=wild type). n=10 cells/experiment were analyzed. Analysis of variance one way (ANOVA) p=1.55e-7; Tukey's post hoc test ***p<0.0005; NS not significant.E. EATR assay in chondrocytes with indicated genotypes (ctrl=wild type) showing % of cells with acidified ER measured by FACS. FGF18 was used at 50 ng/mL overnight. Mean +/- standard error of the mean (sem) of N=4 biological replicates. Analysis of variance one way (ANOVA) p<0.0001: Tukey's post hoc test ***p<0.0005; *p<0.05; NS not significant.F,G Data plots show quantification of mCherry+ vesicles/cell (autolysosomes) (F) and mCherry+/GFP+ vesicles/cell (autophagosomes) (G) in wild type (ctrl) and TFEB;3KO cells treated with vehicle (veh) or FGF18. N=3 independent experiments. Mean +/- standard error of the mean (sem) of N=24 (wild type treated with 5%ABS, veh), N=30 (wild type treated with FGF18), N=27 (TFEB;3KO veh), N= 33 (TFEB;3KO FGF18) cells. Student un-paired T-Test ***p<0.0005; **p<0.005.H. ChIP analysis of TFEB binding to Fam134b DNA in RCS cells transfected with TFEB-3XFLAG. Numbers in the CLEAR site (yellow box) refer to the distance [in base pairs] from the transcriptional start site (+1) of Fam134b-2 gene. Immunoprecipitated DNA was normalized to the input and plotted as relative enrichment over a mock control. Bar graph shows fold change enrichment; mean +/- standard error of the mean (sem) of N=3 independent experiments. Student un-paired T-Test **p<0.005.I-J. Luciferase assays in RCS chondrocytes using as promoter a 0,7kb genomic Fam134b DNA fragment containing a wild type (FAM134B-WT) or a deleted (FAM134B-mut) version of the CLEAR site. TFEB plasmid transfection amount and FGF18 (50 ng/mL for 16h) treatments are indicated. Mean +/- standard error of the mean (sem) of N=3 biological replicates. Student paired T-test *p<0.05; **p<0.005.K. Luciferase activity in wild type (ctrl) and TFEB;3KO RCS chondrocytes with indicated genotypes expressing the indicated Fam134b luciferase report plasmids and treated with FGF18 overnight (50 ng/mL) where indicated. Mean +/- standard error of the mean (sem) of N=5 biological replicates. Analysis of variance one way (ANOVA) p<0.0001: Sidak's multiple comparison test ***p<0.0005; NS not significant.


Figure 6A . qRT - PCR analysis of Fam134b isoforms in mock , TFEB S142A : S211A and TFE3 S246A : S312A over - expressing U2OS cells . Values were normalized to Hprt gene and expressed as fold change values relative to mock . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates . Student ' s un - paired T - test * p < 0 . 05 ; NS not significant . B . Western blot analysis of Fam134b isoforms in mock , TFEB S142A : S211A and TFE3 S246A : S312A over - expressing U2OS cells showing induction of FAM134B - 2 , but not of FAM134B - 1 isoform by TFEB / TFE3 . Filamin A was used as loading control . Blot is representative of N = 3 independent experiments . C , D . Co - immunofluorescence staining of ER ( CLIMP - 63 , green ) and lysosomes ( TMEM192 - HA , red ) in control and Sh - FAM134B U2OS cells over - expressing TFEB S142A : S211A - GFP ( purple ) . BaFA1 was used at 100 nM for 4h . Scale bar 15 μm and 2 μm ( higher magnification boxes ) ( C ) . In ( D ) , Quantification of CLIMP63 fluorescence in TMEM192 - HA decorated lysosomes . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates . N = 21 ( vehicle ctrl ) , N = 33 ( TFEB S142A : S211A - GFP ctrl ) , N = 21 ( vehicle shFAM134B ) , N = 30 ( TFEB S142A : S211A - GFP shFAM134B ) cells were analysed . Student ' s un - paired T - test * * * p < 0 . 0005 ; NS not significant . E , F . Co - immunofluorescence staining of ER ( CLIMP - 63 , green ) and lysosomes ( Lamp1 , red ) in WT and Fam134bKO MEF cells over - expressing TFEB S142A : S211A - GFP ( purple ) . BaFA1 was used at 100 nM for 4h . Scale bar 15 μm and 2 μm ( higher magnification boxes ) ( E ) . In ( F ) , Quantification of CLIMP63 fluorescence in Lamp1 decorated lysosomes . Mean + / - standard error of the mean ( sem ) of N = 3 biological replicates . N = 20 ( vehicle ctrl ) , N = 22 ( TFEB S142A : S211A - GFP ctrl ) , N = 21 ( vehicle Fam134bKO ) , N = 20 ( TFEB S142A : S211A - GFP Fam134bKO ) cells were analysed . Student ' s un - paired T - test * * * p < 0 . 0005 ; NS not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. qRT-PCR analysis of Fam134b isoforms in mock, TFEB S142A:S211A and TFE3 S246A:S312A over-expressing U2OS cells. Values were normalized to Hprt gene and expressed as fold change values relative to mock. Mean +/- standard error of the mean (sem) of N=3 biological replicates. Student's un-paired T-test *p<0.05; NS not significant.B. Western blot analysis of Fam134b isoforms in mock, TFEB S142A:S211A and TFE3 S246A:S312A over-expressing U2OS cells showing induction of FAM134B-2, but not of FAM134B-1 isoform by TFEB/TFE3. Filamin A was used as loading control. Blot is representative of N=3 independent experiments.C,D. Co-immunofluorescence staining of ER (CLIMP-63, green) and lysosomes (TMEM192-HA, red) in control and Sh-FAM134B U2OS cells over-expressing TFEB S142A:S211A-GFP (purple). BaFA1 was used at 100 nM for 4h. Scale bar 15 μm and 2 μm (higher magnification boxes) (C). In (D), Quantification of CLIMP63 fluorescence in TMEM192-HA decorated lysosomes. Mean +/- standard error of the mean (sem) of N=3 biological replicates. N= 21 (vehicle ctrl), N=33 (TFEB S142A:S211A-GFP ctrl), N=21 (vehicle shFAM134B), N= 30 (TFEB S142A:S211A-GFP shFAM134B) cells were analysed. Student's un-paired T-test ***p<0.0005; NS not significant.E,F. Co-immunofluorescence staining of ER (CLIMP-63, green) and lysosomes (Lamp1, red) in WT and Fam134bKO MEF cells over-expressing TFEB S142A:S211A-GFP (purple). BaFA1 was used at 100 nM for 4h. Scale bar 15 μm and 2 μm (higher magnification boxes) (E). In (F), Quantification of CLIMP63 fluorescence in Lamp1 decorated lysosomes. Mean +/- standard error of the mean (sem) of N=3 biological replicates. N= 20 (vehicle ctrl), N=22 (TFEB S142A:S211A-GFP ctrl), N=21 (vehicle Fam134bKO), N= 20 (TFEB S142A:S211A-GFP Fam134bKO) cells were analysed. Student's un-paired T-test ***p<0.0005; NS not significant.


Figure 7A , B . Representative images of alcian blue ( cartilage ) and Alizarin red ( bone ) skeletal staining showing growth retardation in Fgfr3 / 4 dKO mice compared to age / sex wild type littermate at post - natal day 30 . B . Femur , tibia and tail details . C . Haematoxylin / Eosin staining of femoral growth plate sections from wild type and Fgfr3 / 4 dKO mice . Higher magnification insets showed a disorganized hypertrophic chondrocyte layer , in Fgfr3 / 4 dKO mice . Scale bar 60 μm . D . Western blot analysis of IRS1 , p - P70S6K ( T389 ) , P70S6K , p - JNK ( Y185 ) , JNK1 / 2 proteins in growth plate lysates of mice with indicated genotypes . N = 3 mice / genotype . β - actin was used as loading control . Bar graph shows quantification . Mean + / - standard error of the mean ( sem ) . Student ' s un - paired T - test * p < 0 . 05 . E . Western blot analysis of LC3 and Lamp1 proteins from growth plate lysates of mice with indicated genotypes . β - actin was used as loading control . N = 3 mice / genotype were analyzed . Bar graph shows quantification . Mean + / - standard error of the mean ( sem ) . Student ' s un - paired T - test * p < 0 . 05 . F . qRT - PCR analysis of Fam134b - 2 expression from growth plate of mice with indicated genotypes . N = 8 ( wt mice ) and N = 9 ( Fgfr3 / 4 dKO mice ) were analyzed . Values were normalized to Hprt gene and expressed as fold change relative to control . Mean + / - standard error of the mean ( sem ) . Student ' s un - paired T - test * p < 0 . 05 . G . Western blot analysis of Fam134b - 2 protein from growth plate lysates of mice with indicated genotypes . β - actin was used as loading control . N = 6 ( wt mice ) and N = 4 ( Fgfr3 / 4 dKO mice ) were analyzed . Bar graph showed quantification of Fam134b - 2 normalized to β - actin . Mean + / - standard error of the mean ( sem ) . Student ' s un - paired T - test * p < 0 . 05 . H . Representative immunofluorescence staining of CLIMP - 63 of femur growth plate sections from mice with indicated genotypes . Scale bar 10μm . Insets showed increased CLIMP - 63 staining in Fgfr3 / 4 dKO mice . Scale bar 5 μm . Hypert = hypertrophic chondrocytes ; pre - hypert = pre - hypertrophic chondrocytes ; prolif = proliferating chondrocytes . I . Representative TEM images of growth plate chondrocytes from mice with indicated genotypes . Arrows indicated the ER . N = nucleus . Scale bar 1μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A,B. Representative images of alcian blue (cartilage) and Alizarin red (bone) skeletal staining showing growth retardation in Fgfr3/4 dKO mice compared to age/sex wild type littermate at post-natal day 30. B. Femur, tibia and tail details.C. Haematoxylin/Eosin staining of femoral growth plate sections from wild type and Fgfr3/4 dKO mice. Higher magnification insets showed a disorganized hypertrophic chondrocyte layer, in Fgfr3/4 dKO mice. Scale bar 60 μm.D. Western blot analysis of IRS1, p-P70S6K (T389), P70S6K, p-JNK (Y185), JNK1/2 proteins in growth plate lysates of mice with indicated genotypes. N=3 mice/genotype. β-actin was used as loading control. Bar graph shows quantification. Mean +/- standard error of the mean (sem). Student's un-paired T-test *p<0.05.E. Western blot analysis of LC3 and Lamp1 proteins from growth plate lysates of mice with indicated genotypes. β-actin was used as loading control. N=3 mice/genotype were analyzed. Bar graph shows quantification. Mean +/- standard error of the mean (sem). Student's un-paired T-test *p<0.05.F. qRT-PCR analysis of Fam134b-2 expression from growth plate of mice with indicated genotypes. N=8 (wt mice) and N=9 (Fgfr3/4 dKO mice) were analyzed. Values were normalized to Hprt gene and expressed as fold change relative to control. Mean +/- standard error of the mean (sem). Student's un-paired T-test *p<0.05.G. Western blot analysis of Fam134b-2 protein from growth plate lysates of mice with indicated genotypes. β-actin was used as loading control. N=6 (wt mice) and N=4 (Fgfr3/4 dKO mice) were analyzed. Bar graph showed quantification of Fam134b-2 normalized to β-actin. Mean +/- standard error of the mean (sem). Student's un-paired T-test *p<0.05.H. Representative immunofluorescence staining of CLIMP-63 of femur growth plate sections from mice with indicated genotypes. Scale bar 10μm. Insets showed increased CLIMP-63 staining in Fgfr3/4 dKO mice. Scale bar 5 μm. Hypert= hypertrophic chondrocytes; pre-hypert= pre-hypertrophic chondrocytes; prolif = proliferating chondrocytes.I. Representative TEM images of growth plate chondrocytes from mice with indicated genotypes. Arrows indicated the ER. N= nucleus. Scale bar 1μm.


Figure 8A . Scatter plot of cellular compartments and biological processes regulated by FGF18 in a Fam134b - dependent manner . Score : Student ' s T - test difference between wild type and Fam134b ∆ LIR cells . N = 3 biological replicates / treatment / genotype were analysed . FDR < 0 . 05 . B . Tandem mass tag secretome analysis of chondrocytes with indicated genotypes and treatments . Fam134b - dependent secreted proteins are shown N = 3 biological replicates / treatment / genotype were analysed . FDR < 0 . 05 . C . qRT - PCR analysis of Fam134b and Lamp1 from medaka fish embryos treated with FGF18 ( 50 ng / mL ) for 24h . Values were normalized to Hprt gene and expressed as fold change relative to untreated fish . N = 5 biological replicates . Mean + / - standard error of the mean ( sem ) . Paired Student ' s T - test * * p < 0 . 005 ; * p < 0 . 05 . D . Co - immunofluorescence staining of Lamp1 ( lysosome ) and PDI ( ER ) in medaka fish chondrocytes with indicated genotypes treated with FGF18 ( 50 ng / mL for 24h ) . Insets showed details of colocalization of Lamp1 and PDI . Wheat germ agglutinin ( WGA ) was used to stain cartilage matrix . Representative images of N = 3 fish / genotype / treatment . Scale bars 10 μm . Boxed area were zoomed and single and merged channels are shown . Scale bars 2 μm . E . Quantification of PDI fluorescence intensity into Lamp1 positive lysosomes . Mean + / - standard error of the mean ( sem ) of N = 10 cells / experiment were analyzed . Analysis of variance one way ( ANOVA ) p < 0 . 001 ; Sidak ' s multiple comparison test * * * p < 0 . 0005 ; NS not significant . F . Western blot analysis of type II procollagen from a pool of medaka fish embryos with indicated genotypes , showing procollagen accumulation in Fam134bmo fish . β - actin was used as loading control . Bar graph shows quantification of type II procollagen normalized to β - actin . N = 3 biological replicates . Mean + / - standard error of the mean ( sem ) . Student ' s un - paired T - test * p < 0 . 05 . G . Representative TEM images of medaka fish chondrocytes . Arrows indicated the ER . Insets show ER enlargement in Fam134bmo chondrocyte . N = 3 fish / genotype were analyzed . Bar graph shows number of ER enlargement per cell in medaka fish with indicated genotypes . N = 29 cells ( Fam134bwt ) and N = 37 cells ( Fam134bmo ) were analyzed . Mean + / - standard error of the mean ( sem ) . Student ' s un - paired T - test * * * p < 0 . 0005 . Scale bar 500 nm . N = nucleus , ER = Endoplasmic Reticulum . H . Lateral ( top ) and ventral ( bottom ) projections of medaka fish embryos with indicated genotypes . Scale bar 1mm . Bar graphs show quantification of total length and head size of medaka fish model of Fam134bmo expressed as % relative to the scramble . Mean + / - standard error of the mean ( sem ) of at least n = 8 fish / genotype . Student un - paired T - Test * p < 0 . 05 . Alcian blue ( cartilage ) ( I ) staining of scramble and Fam134bmo medaka fish . Graph shows quantification of Ethmoid plate ( EP ) , Palatoquadrate ( PQ ) , Ceratohyal ( CH ) , Paired Prootics ( PO ) , Ceretobranchials 1 to 5 ( CB1 to CB5 ) cartilage length ( I ) in Fam134bmo and scramble fish . Values were expressed as % relative to the scramble ( 100 % red dotted line ) . Mean + / - standard error of the mean ( sem ) of n = 9 fish / genotype . Student unpaired T - Test * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 0005 . NS not significant . Alizarin Red ( bone ) ( J ) staining of scramble and Fam134bmo medaka fish . Graph shows quantification of Ethmoid plate ( EP ) , Palatoquadrate ( PQ ) , Ceratohyal ( CH ) , Paired Prootics ( PO ) , Ceretobranchials 1 to 5 ( CB1 to CB5 ) bone mineralization ( J ) in Fam134bmo and scramble fish . Values were expressed as % relative to the scramble ( 100 % red dotted line ) . Mean + / - standard error of the mean ( sem ) of n = 9 fish / genotype . Student unpaired T - Test * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 0005 . NS not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A. Scatter plot of cellular compartments and biological processes regulated by FGF18 in a Fam134b-dependent manner. Score: Student's T-test difference between wild type and Fam134b∆LIR cells. N=3 biological replicates/treatment/genotype were analysed. FDR <0.05.B. Tandem mass tag secretome analysis of chondrocytes with indicated genotypes and treatments. Fam134b-dependent secreted proteins are shown N=3 biological replicates/treatment/genotype were analysed. FDR <0.05.C. qRT-PCR analysis of Fam134b and Lamp1 from medaka fish embryos treated with FGF18 (50 ng/mL) for 24h. Values were normalized to Hprt gene and expressed as fold change relative to untreated fish. N= 5 biological replicates. Mean +/- standard error of the mean (sem). Paired Student's T-test **p<0.005; *p<0.05.D. Co-immunofluorescence staining of Lamp1 (lysosome) and PDI (ER) in medaka fish chondrocytes with indicated genotypes treated with FGF18 (50 ng/mL for 24h). Insets showed details of colocalization of Lamp1 and PDI. Wheat germ agglutinin (WGA) was used to stain cartilage matrix. Representative images of N=3 fish/genotype/treatment. Scale bars 10 μm. Boxed area were zoomed and single and merged channels are shown. Scale bars 2 μm.E. Quantification of PDI fluorescence intensity into Lamp1 positive lysosomes. Mean +/- standard error of the mean (sem) of N=10 cells/experiment were analyzed. Analysis of variance one way (ANOVA) p<0.001; Sidak's multiple comparison test ***p<0.0005; NS not significant.F. Western blot analysis of type II procollagen from a pool of medaka fish embryos with indicated genotypes, showing procollagen accumulation in Fam134bmo fish. β-actin was used as loading control. Bar graph shows quantification of type II procollagen normalized to β-actin. N=3 biological replicates. Mean +/- standard error of the mean (sem). Student's un-paired T-test *p<0.05.G. Representative TEM images of medaka fish chondrocytes. Arrows indicated the ER. Insets show ER enlargement in Fam134bmo chondrocyte. N=3 fish/genotype were analyzed. Bar graph shows number of ER enlargement per cell in medaka fish with indicated genotypes. N=29 cells (Fam134bwt) and N=37 cells (Fam134bmo) were analyzed. Mean +/- standard error of the mean (sem). Student's un-paired T-test ***p<0.0005. Scale bar 500 nm. N=nucleus, ER= Endoplasmic Reticulum.H. Lateral (top) and ventral (bottom) projections of medaka fish embryos with indicated genotypes. Scale bar 1mm. Bar graphs show quantification of total length and head size of medaka fish model of Fam134bmo expressed as % relative to the scramble. Mean +/- standard error of the mean (sem) of at least n=8 fish/genotype. Student un-paired T-Test *p<0.05.Alcian blue (cartilage) (I) staining of scramble and Fam134bmo medaka fish. Graph shows quantification of Ethmoid plate (EP), Palatoquadrate (PQ), Ceratohyal (CH), Paired Prootics (PO), Ceretobranchials 1 to 5 (CB1 to CB5) cartilage length (I) in Fam134bmo and scramble fish. Values were expressed as % relative to the scramble (100% red dotted line). Mean +/- standard error of the mean (sem) of n=9 fish/genotype. Student unpaired T-Test *p<0.05; **p<0.005; ***p<0.0005. NS not significant.Alizarin Red (bone) (J) staining of scramble and Fam134bmo medaka fish. Graph shows quantification of Ethmoid plate (EP), Palatoquadrate (PQ), Ceratohyal (CH), Paired Prootics (PO), Ceretobranchials 1 to 5 (CB1 to CB5) bone mineralization (J) in Fam134bmo and scramble fish. Values were expressed as % relative to the scramble (100% red dotted line). Mean +/- standard error of the mean (sem) of n=9 fish/genotype. Student unpaired T-Test *p<0.05; **p<0.005; ***p<0.0005. NS not significant.


Figure 1 ( D ) Immunofluorescence of neurons expressing BioID2 - tau or BioID2 ( myc ) or non - transduced control cells . Cells were incubated in biotin medium ( 50 μM ) or vehicle medium for 30 h prior to fixation and probed for biotin ( streptavidin , SA ) and cell nuclei ( DAPI ) ( n = 3 biological replicates ) . BioID2 - tau and incorporated biotin co - localise in axons and somata . Scale bar , 25 μm .

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Figure 1(D) Immunofluorescence of neurons expressing BioID2-tau or BioID2 (myc) or non-transduced control cells. Cells were incubated in biotin medium (50 μM) or vehicle medium for 30 h prior to fixation and probed for biotin (streptavidin, SA) and cell nuclei (DAPI) (n = 3 biological replicates). BioID2-tau and incorporated biotin co-localise in axons and somata. Scale bar, 25 μm.


Figure 2 ( B ) Immunoblot of combined brain lysates from mice ( P35 ) expressing BioID2 - tau or BioID2 control ( n = 4 biological replicates ) probed for myc - tagged BioID2 and BioID2 - tau . Controls : lysates from ( 1 ) BioID2 - or ( 2 ) BioID2 - tau - expressing and ( 3 ) non - transduced mouse brain . Loading , glyceraldehyde - 3 - phosphate dehydrogenase ( Gapdh ) . * , unspecific band . Relative quantification confirmed similar expression levels of BioID2 and BioID2 - tau . Means ± S . E . M . ns , not significant ( Student ' s t - test ) ( C ) Immunofluorescence of P35 mouse brain expressing BioID2 - tau . Hippocampal Cornu ammonis 1 ( CA1 ) region was probed for BioID2 - tau ( myc ) , for incorporated biotin ( streptavidin , SA ) and for cell nuclei ( DAPI ) ( n = 3 - 5 biological replicates ) . Scale bar , 50 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) Immunoblot of combined brain lysates from mice (P35) expressing BioID2-tau or BioID2 control (n = 4 biological replicates) probed for myc-tagged BioID2 and BioID2-tau. Controls: lysates from (1) BioID2- or (2) BioID2-tau-expressing and (3) non-transduced mouse brain. Loading, glyceraldehyde-3-phosphate dehydrogenase (Gapdh). *, unspecific band. Relative quantification confirmed similar expression levels of BioID2 and BioID2-tau. Means ± S.E.M. ns, not significant (Student's t-test)(C) Immunofluorescence of P35 mouse brain expressing BioID2-tau. Hippocampal Cornu ammonis 1 (CA1) region was probed for BioID2-tau (myc), for incorporated biotin (streptavidin, SA) and for cell nuclei (DAPI) (n = 3-5 biological replicates). Scale bar, 50 μm.


Figure 4 ( A ) Confocal immunofluorescence for endogenous tau and NSF in primary murine hippocampal neurons ( DIV15 ) . Colocalization of tau and NSF is apparent in perinuclear compartments ( arrows ) and neurites ( insets i - iii ) . Scale bar , 25 μm . Inset of magnified neurite with partial overlap of tau and NSF signals ( arrowheads ) . Scale bar , 3 μm . Co - distribution ( R Ch1 - Ch2 ) are indicated . ( B - C ) Co - immunoprecipitation ( Co - IP ) of tau and NSF from mouse brain . ( B ) Endogenous and transgenic tau were immunoprecipitated from mouse brain lysates of tau - / - , ALZ17tg or ALZ17non - tg mice using anti - tau antibody followed by immunoblot probed for NSF , tau and α - actin . Endogenous NSF was co - immunoprecipitated with transgenic and endogenous tau from ALZ17tg or ALZ17non - tg but not from tau - / - with . GAPDH , input loading control ( n = 3 biological replicates ) . Recombinant tau and NSF served as controls . ( C ) Densitometric quantification of co - IP results in ( B ) . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test . Co - IP of HA - tagged tau with NSF from 293T cells . ( E ) Co - IP of NSF with haemagglutinin ( HA ) - tagged tau variants from cultured cells . HA - tagged tau variants and NSF were expressed in 293T cells followed by immunoprecipitation using HA antibody and immunoblot probed for NSf , tau and GAPDH . ( n = 3 biological replicates ) . ( F ) Microscale thermophoretic ( MST ) measurement of direct tau and NSF interaction . MST binding curve of recombinant hexameric NT - 647 - NHS - labelled NSF ( 27 pM ) and recombinant tau ( 18 mM - 0 . 275 nM ) . Dissociation constant : KD = 146 ± 70 nM ( sigmoidal curve fit , R2 = 0 . 844 ) . ( n = 5 technical replicates ) . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test . ( G ) Inhibition of NSF ATPase activity with increasing concentrations of tau . NSF activity ( 70 nM ) was assessed alone or in the presence of NSF - inhibiting peptide TAT - NSF or control TAT - Scramble peptides ( 140 nM ) ; or co - incubated with recombinant tau protein ( 8 nM , 16 nM , 24 nM ) , or heat - denatured tau ( dTau , 8 nM ) or albumin ( Alb , 16 nM ) . Data were fitted to Michaelis - Menten model to determine rate constants ( R2 = 0 . 904 - 0 . 994 for all conditions ; n = 3 technical replicates ) . ( H ) Rate constants derived from kinetic data in i . ( n = 3 technical replicates ) ATP hydrolysis is similar in NSF and NSF incubated with control peptide TAT - Scr , albumin , or denatured tau , yet lower with increasing concentrations of tau or after addition of NSF - inhibiting peptide TAT - NSF . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Confocal immunofluorescence for endogenous tau and NSF in primary murine hippocampal neurons (DIV15). Colocalization of tau and NSF is apparent in perinuclear compartments (arrows) and neurites (insets i-iii). Scale bar, 25 μm. Inset of magnified neurite with partial overlap of tau and NSF signals (arrowheads). Scale bar, 3 μm. Co-distribution (R Ch1-Ch2) are indicated.(B-C) Co-immunoprecipitation (Co-IP) of tau and NSF from mouse brain. (B) Endogenous and transgenic tau were immunoprecipitated from mouse brain lysates of tau-/-, ALZ17tg or ALZ17non-tg mice using anti-tau antibody followed by immunoblot probed for NSF, tau and α-actin. Endogenous NSF was co-immunoprecipitated with transgenic and endogenous tau from ALZ17tg or ALZ17non-tg but not from tau-/- with. GAPDH, input loading control (n = 3 biological replicates). Recombinant tau and NSF served as controls. (C) Densitometric quantification of co-IP results in (B). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.Co-IP of HA-tagged tau with NSF from 293T cells. (E) Co-IP of NSF with haemagglutinin (HA)-tagged tau variants from cultured cells. HA-tagged tau variants and NSF were expressed in 293T cells followed by immunoprecipitation using HA antibody and immunoblot probed for NSf, tau and GAPDH. (n = 3 biological replicates).(F) Microscale thermophoretic (MST) measurement of direct tau and NSF interaction. MST binding curve of recombinant hexameric NT-647-NHS-labelled NSF (27 pM) and recombinant tau (18 mM - 0.275 nM). Dissociation constant: KD = 146 ± 70 nM (sigmoidal curve fit, R2 = 0.844). (n = 5 technical replicates). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.(G) Inhibition of NSF ATPase activity with increasing concentrations of tau. NSF activity (70 nM) was assessed alone or in the presence of NSF-inhibiting peptide TAT-NSF or control TAT-Scramble peptides (140 nM); or co-incubated with recombinant tau protein (8 nM, 16 nM, 24 nM), or heat-denatured tau (dTau, 8 nM) or albumin (Alb, 16 nM). Data were fitted to Michaelis-Menten model to determine rate constants (R2 = 0.904 - 0.994 for all conditions; n = 3 technical replicates).(H) Rate constants derived from kinetic data in i. (n = 3 technical replicates) ATP hydrolysis is similar in NSF and NSF incubated with control peptide TAT-Scr, albumin, or denatured tau, yet lower with increasing concentrations of tau or after addition of NSF-inhibiting peptide TAT-NSF. Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


Figure 5 ( A ) Impaired AMPA - type glutamate receptor surface expression in cultured tau - / - neurons . Cortical tau + / + or tau - / - neurons were surface biotinylated after incubation with non - competitive NMDAR antagonist MK - 801 ( 50 μM , 3 min ) and / or GABAAR antagonist bicuculline ( 30 μM , 10 min ) at 37 ° C . Surface GluA1 levels were detected by immunoblot after with streptavidin ( SA ) enrichment . Input was probed for total cellular GluA1 , NSF , tau and βIII - tubulin . ( n = 3 biological replicates ) Input and SA - enrichment from non - surface biotinylated neurons served as negative controls . ( B ) Reduced neuronal surface GluA1 in tau - / - neurons . Quantification of tau + / + and tau - / - surface GluA1 upon treatment and SA - enrichment shown in ( A ) . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test . ( C ) Cell surface GluA1 and total NSF immunostaining in primary mouse hippocampal neurons ( DIV15 ) . Tau + / + , tau - / - neurons and tau - / - expressing human tau ( tau - / - . AAVtau ) were treated with either vehicle ( Veh ; DMSO ) or MK - 801 ( 50 μM ) for 3 min . Representative confocal images of whole cell ( left ) and neurites ( right ) are shown ( n = 3 biological replicates ) . NSF , surface GluA1 and cell nuclei ( DAPI ) were visualized . Scale bar , 25 μm . ( D - E ) Quantification of neurite surface GluA1 and neurite NSF relative to tau + / + Veh control . Reduced surface GluA1 ( D ) and total NSF ( n = 10 - 12 neurites from 3 biological replicates ) ( E ) localized to neurites in tau - / - neurons treated with MK801 ( n = 21 - 23 neurites from 3 biological replicates ) . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test . ( F ) Changes in secondary dendrite GluA2 expression in neurons ( DIV18 ) upon cLTP / cLTD induction . Tau - / - neurons were transfected with tau at DIV12 ( = tau - / - . tau ) or mock transfected ( tau - / - ) . Cells were stained for GluA2 , dendritic marker Map2 , and NSF at 2 h post - cLTP / cLTD induction or treatment with control artificial cerebrospinal fluid ( aCSF ) . Dendritic GluA2 cluster analysis was based on GluA2 - Map2 binary intensity map . Representative insets from n = 10 - 20 dendrites per condition from 2 mice per genotype . Scale bar , 3 μm . ( G ) Quantification of GluA2 mean intensity relative to Map2 area and normalised to aCSF controls . Secondary dendrite GluA2 increased in tau - expressing ( tau + / + and tau - / - . tau ) neurons upon cLTP induction . ( n = 10 - 19 dendrites from 3 biological replicates ) . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test . ( H ) Representative immunostaining for whole - cell NSF and GluA2 in tau + / + , tau - / - and tau - transfected tau - / - ( tau - / - . tau ) neurons at 2 h post - cLTP / cLTD induction or treatment with control artificial cerebrospinal fluid ( aCSF ) . ( n = 3 - 6 biological replicates per condition with 10 - 20 cells each ) . Scale bar , 10 μm . Arrows indicate areas of marked co - distribution . ( I ) Co - localisation analysis of NSF and GluA2 upon chemical LTP / LTD induction . Note significantly higher proportional co - distribution of NSF and GluA2 in tau - expressing ( tau + / + and tau - / - . tau ) cells upon cLTP induction . ( n = 3 - 6 cells from 3 biological replicates ) . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Impaired AMPA-type glutamate receptor surface expression in cultured tau-/- neurons. Cortical tau+/+ or tau-/- neurons were surface biotinylated after incubation with non-competitive NMDAR antagonist MK-801 (50 μM, 3 min) and/or GABAAR antagonist bicuculline (30 μM, 10 min) at 37°C. Surface GluA1 levels were detected by immunoblot after with streptavidin (SA) enrichment. Input was probed for total cellular GluA1, NSF, tau and βIII-tubulin. (n = 3 biological replicates) Input and SA-enrichment from non-surface biotinylated neurons served as negative controls. (B) Reduced neuronal surface GluA1 in tau-/- neurons. Quantification of tau+/+ and tau-/- surface GluA1 upon treatment and SA-enrichment shown in (A). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test. (C) Cell surface GluA1 and total NSF immunostaining in primary mouse hippocampal neurons (DIV15). Tau+/+, tau-/- neurons and tau-/- expressing human tau (tau-/-.AAVtau) were treated with either vehicle (Veh; DMSO) or MK-801 (50 μM) for 3 min. Representative confocal images of whole cell (left) and neurites (right) are shown (n = 3 biological replicates). NSF, surface GluA1 and cell nuclei (DAPI) were visualized. Scale bar, 25 μm. (D-E) Quantification of neurite surface GluA1 and neurite NSF relative to tau+/+ Veh control. Reduced surface GluA1 (D) and total NSF (n = 10-12 neurites from 3 biological replicates) (E) localized to neurites in tau-/- neurons treated with MK801 (n = 21-23 neurites from 3 biological replicates). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test. (F) Changes in secondary dendrite GluA2 expression in neurons (DIV18) upon cLTP/cLTD induction. Tau-/- neurons were transfected with tau at DIV12 (= tau-/-.tau) or mock transfected (tau-/-). Cells were stained for GluA2, dendritic marker Map2, and NSF at 2 h post-cLTP/cLTD induction or treatment with control artificial cerebrospinal fluid (aCSF). Dendritic GluA2 cluster analysis was based on GluA2-Map2 binary intensity map. Representative insets from n = 10-20 dendrites per condition from 2 mice per genotype. Scale bar, 3 μm. (G) Quantification of GluA2 mean intensity relative to Map2 area and normalised to aCSF controls. Secondary dendrite GluA2 increased in tau-expressing (tau+/+ and tau-/-.tau) neurons upon cLTP induction. (n = 10-19 dendrites from 3 biological replicates). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test. (H) Representative immunostaining for whole-cell NSF and GluA2 in tau+/+, tau-/- and tau-transfected tau-/- (tau-/-.tau) neurons at 2 h post-cLTP/cLTD induction or treatment with control artificial cerebrospinal fluid (aCSF). (n = 3-6 biological replicates per condition with 10-20 cells each). Scale bar, 10 μm. Arrows indicate areas of marked co-distribution. (I) Co-localisation analysis of NSF and GluA2 upon chemical LTP/LTD induction. Note significantly higher proportional co-distribution of NSF and GluA2 in tau-expressing (tau+/+ and tau-/-.tau) cells upon cLTP induction. (n = 3-6 cells from 3 biological replicates). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


Figure 6 ( B ) Examples of sagittal sections of tau - / - and AAV . syn - hTau - injected tau - / - ( tau - / - . AAVtau ) mice showing localized AAV - mediated expression of eGFP or tau and position of infusion cannulas ending in the hippocampus ( dashed line ) . GluA1 , immunolabelling for GluA1 AMPA receptor subunit . Scale bar , 50 μm . ( C ) Cued fear conditioning memory in tau + / + , tau - / - and tau - / - . AAVtau mice upon bilateral infusion of TAT - NSF or TAT - Scr . Due to infusion of Tat - fusion peptides occurring under brief isoflurane anaesthesia , a general anaesthesia ( GA ) control is included . Note that infusion of TAT - NSF lowers memory performance to comparable levels in tau + / + , tau - / - and tau - / - . AAVtau mice . ( n = 2 - 5 biological replicates ) Data information : Values are mean ± 95 % confidence intervals . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test . ( D ) Novel object preference ( fold preference over familiar object ) in tau + / + , tau - / - and tau - / - . AAVtau mice upon bilateral infusion of TAT - NSF or TAT - Scr ( n = 2 - 5 biological replicates ) . Due to infusion of Tat - fusion peptides occurring under brief isoflurane anaesthesia , a general anaesthesia ( GA ) control is included ( n = 2 for GA ) . Note that infusion of TAT - NSF lowers object discrimination to comparable levels in tau + / + , tau - / - and tau - / - . AAVtau mice . Data information : Values are mean ± 95 % confidence intervals . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test . ( E ) ATPase assay of recombinant NSF upon incubation with tau coimmunoprecipitates ( tau5 ) from cortical lysates of indicated genotypes , including from tau - transgenic mice ( ALZ17 , Tau58 / 2 ) ( n = 5 - 8 biological replicates ) . IgG , control immunoprecipitate with non - specific IgG from tau + / + lysate . Data information : Values are mean ± 95 % confidence intervals . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test . ( F ) ATPase assay of recombinant NSF upon incubation with tau coimmunoprecipitates ( tau5 ) from brain lysates of healthy controls ( HC ) , mild cognitive impairment ( MCI ) , and Alzheimer ' s disease ( AD ) ( n = 3 - 4 biological replicates ) . IgG , control immunoprecipitate . Data information : Values are mean ± 95 % confidence intervals . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(B) Examples of sagittal sections of tau-/- and AAV.syn-hTau-injected tau-/- (tau-/-.AAVtau) mice showing localized AAV-mediated expression of eGFP or tau and position of infusion cannulas ending in the hippocampus (dashed line). GluA1, immunolabelling for GluA1 AMPA receptor subunit. Scale bar, 50 μm.(C) Cued fear conditioning memory in tau+/+, tau-/- and tau-/-.AAVtau mice upon bilateral infusion of TAT-NSF or TAT-Scr. Due to infusion of Tat-fusion peptides occurring under brief isoflurane anaesthesia, a general anaesthesia (GA) control is included. Note that infusion of TAT-NSF lowers memory performance to comparable levels in tau+/+, tau-/- and tau-/-.AAVtau mice. (n = 2-5 biological replicates) Data information: Values are mean ± 95% confidence intervals. Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.(D) Novel object preference (fold preference over familiar object) in tau+/+, tau-/- and tau-/-.AAVtau mice upon bilateral infusion of TAT-NSF or TAT-Scr (n = 2-5 biological replicates). Due to infusion of Tat-fusion peptides occurring under brief isoflurane anaesthesia, a general anaesthesia (GA) control is included (n = 2 for GA). Note that infusion of TAT-NSF lowers object discrimination to comparable levels in tau+/+, tau-/- and tau-/-.AAVtau mice. Data information: Values are mean ± 95% confidence intervals. Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.(E) ATPase assay of recombinant NSF upon incubation with tau coimmunoprecipitates (tau5) from cortical lysates of indicated genotypes, including from tau-transgenic mice (ALZ17, Tau58/2) (n = 5-8 biological replicates). IgG, control immunoprecipitate with non-specific IgG from tau+/+ lysate. Data information: Values are mean ± 95% confidence intervals. Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.(F) ATPase assay of recombinant NSF upon incubation with tau coimmunoprecipitates (tau5) from brain lysates of healthy controls (HC), mild cognitive impairment (MCI), and Alzheimer's disease (AD) (n = 3-4 biological replicates). IgG, control immunoprecipitate. Data information: Values are mean ± 95% confidence intervals. Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


Figure 1 ( D ) NIH 3T3 cells transfected by equal amounts of plasmid fused with gfp ( without ATG ) to the sequence from 5 ' cap of pri - miR - 31 to ATG site or which mutated to ATT of the sORF . Cells were selected by G418 for 2 days . Analysis of the GFP expression by confocal microscopy or flow cytometry , scale bar 100 μm . ( E ) Immunoblot analysis of GFP and miPEP31 in NIH 3T3 cells transfected by pEGFP - N1 - miPEP31 or pEGFP - N1 , the red arrow indicate predicted size of miPEP31 - GFP band . ( F ) Flow - fish to detect the localization of pri - miR - 31 . The location of pri - miR - 31 was analyzed by confocal microscopy . Scale bar 5 μm . ( G ) Immunoblot analysis of multiple mouse tissues with a specific antibody against miPEP31 . Values are expressed as fold changes relative to controls and normalized to GAPDH , the red arrow indicate predicted size of miPEP31 . ( H ) Immunoblot analysis of spleen , lymph node and thymus from WT or miR - 31 - / - mice with miPEP31 antibody , the red arrow indicate predicted size of miPEP31 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(D) NIH 3T3 cells transfected by equal amounts of plasmid fused with gfp (without ATG) to the sequence from 5' cap of pri-miR-31 to ATG site or which mutated to ATT of the sORF. Cells were selected by G418 for 2 days. Analysis of the GFP expression by confocal microscopy or flow cytometry, scale bar 100 μm.(E) Immunoblot analysis of GFP and miPEP31 in NIH 3T3 cells transfected by pEGFP-N1-miPEP31 or pEGFP-N1, the red arrow indicate predicted size of miPEP31-GFP band.(F) Flow-fish to detect the localization of pri-miR-31. The location of pri-miR-31 was analyzed by confocal microscopy. Scale bar 5 μm.(G) Immunoblot analysis of multiple mouse tissues with a specific antibody against miPEP31. Values are expressed as fold changes relative to controls and normalized to GAPDH, the red arrow indicate predicted size of miPEP31.(H) Immunoblot analysis of spleen, lymph node and thymus from WT or miR-31-/- mice with miPEP31 antibody, the red arrow indicate predicted size of miPEP31.