( B ) Violin plots of Vero - E6 cells labeled for Spike protein . The Multi - wavelength Cell Scoring algorithm in MetaXpress was used to determine the integrated fluorescent signal in individual cells as a measure of the amount of Spike protein within each cell . The plots show the population distribution of the integrated signal for all of the treatments . The Boxes in the plot show the interquartile range ( IQR ) with the top and bottom edges marking the 75th and 25th percentiles , respectively . The horizontal line in the box is the median value , and the whiskers are defined to be 1 . 5 IQR . The ordinate is a log scale . The effect of the treatment is assessed quantitatively by changes in the median signal level , and qualitatively by observing changes in the modes . The dashed line is 3 standard deviations above the mean signal in the Mock samples and is used as a cut - off to quantify the number of cells that are positive or negative for the Spike signal . The statistics table below the plots shows the number of cells counted in each treatment group , and the median of the population .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Violin plots of Vero-E6 cells labeled for Spike protein. The Multi-wavelength Cell Scoring algorithm in MetaXpress was used to determine the integrated fluorescent signal in individual cells as a measure of the amount of Spike protein within each cell. The plots show the population distribution of the integrated signal for all of the treatments. The Boxes in the plot show the interquartile range (IQR) with the top and bottom edges marking the 75th and 25th percentiles, respectively. The horizontal line in the box is the median value, and the whiskers are defined to be 1.5 IQR. The ordinate is a log scale. The effect of the treatment is assessed quantitatively by changes in the median signal level, and qualitatively by observing changes in the modes. The dashed line is 3 standard deviations above the mean signal in the Mock samples and is used as a cut-off to quantify the number of cells that are positive or negative for the Spike signal. The statistics table below the plots shows the number of cells counted in each treatment group, and the median of the population.


HEK293 acceptor cells transfected with or without ACE2 and TMPRSS2 were seeded in 384 well plates , pretreated with 7 - point gradients of test compounds for 1 - 2 h , and co - cultured for 4 hours with HEK293 donor cells expressing SARS - CoV - 2 spike and GFP , or donor cells expressing GFP only ( no spike ) . Images of GFP - positive objects were acquired on a confocal high - content imager and analyzed for syncytia formation and integrated GFP area ( total GFP ) as a measure of cytotoxicity , using a CNT algorithm as described in the Materials and Methods . Data are the aggregate of 8 independent biological repeats ; where errors are shown they represent SD from matching concentrations in at least three experiments .

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

HEK293 acceptor cells transfected with or without ACE2 and TMPRSS2 were seeded in 384 well plates, pretreated with 7-point gradients of test compounds for 1-2 h, and co-cultured for 4 hours with HEK293 donor cells expressing SARS-CoV-2 spike and GFP, or donor cells expressing GFP only (no spike). Images of GFP-positive objects were acquired on a confocal high-content imager and analyzed for syncytia formation and integrated GFP area (total GFP) as a measure of cytotoxicity, using a CNT algorithm as described in the Materials and Methods. Data are the aggregate of 8 independent biological repeats; where errors are shown they represent SD from matching concentrations in at least three experiments.


Peptide array dot blot analysis to identify LIR motifs in the indicated SNARE proteins . Amino acids for the identified LIRs from positive signals are marked on each SNARE . Amino acids are denoted as amino acid single letter codes in the blot .

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

Peptide array dot blot analysis to identify LIR motifs in the indicated SNARE proteins. Amino acids for the identified LIRs from positive signals are marked on each SNARE. Amino acids are denoted as amino acid single letter codes in the blot.


Co - immunoprecipitation ( Co - IP ) analysis of interactions between FLAG - tagged Stx16 / Vti1a / Stx6 and EGFP - tagged LC3B or GABARAP overexpressed in HEK293T cells . * indicates mouse IgG heavy chain of precipitated mouse - anti - FLAG antibody .

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Co-immunoprecipitation (Co-IP) analysis of interactions between FLAG-tagged Stx16/Vti1a/Stx6 and EGFP-tagged LC3B or GABARAP overexpressed in HEK293T cells. * indicates mouse IgG heavy chain of precipitated mouse-anti-FLAG antibody.


GST pull - down analysis of interactions between radiolabeled Myc - Stx16 and GST - tagged mAtg8 proteins .

GST pull-down analysis of interactions between radiolabeled Myc-Stx16 and GST-tagged mAtg8 proteins.


GST pull - down analysis of interactions between wild type ( WT ) or LIR - mutant ( L219A / V222A ) Stx16 and GST - tagged LC3C or GABARAP . Lower panel shows percentages of WT or LIR - mutant Stx16 bound to GST - LC3C or GST - GABARAP . Data shown as means ± SEM of precipitated Stx16 , n = 3 .

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

GST pull-down analysis of interactions between wild type (WT) or LIR-mutant (L219A/V222A) Stx16 and GST-tagged LC3C or GABARAP. Lower panel shows percentages of WT or LIR-mutant Stx16 bound to GST-LC3C or GST-GABARAP. Data shown as means ± SEM of precipitated Stx16, n = 3.


GST pull - down analysis of interactions between WT or different types of LIR - mutant Stx16 and GST - tagged LC3C or GABARAP . Lower panel shows percentages of WT or LIR - mutant Stx16 bound to GST - LC3C or GST - GABARAP .

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GST pull-down analysis of interactions between WT or different types of LIR-mutant Stx16 and GST-tagged LC3C or GABARAP. Lower panel shows percentages of WT or LIR-mutant Stx16 bound to GST-LC3C or GST-GABARAP.


GST pull - down analysis of interactions between radiolabeled Myc - Stx16 and WT or LDS - mutant GABARAP ( GABARAP - Y49A and - Y49A / F104A ) . Lower panel shows percentages of Stx16 bound to WT or LDS - mutant GABARAP . Data shown as means ± SEM of precipitated Stx16 , n = 3 .

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

GST pull-down analysis of interactions between radiolabeled Myc-Stx16 and WT or LDS-mutant GABARAP (GABARAP-Y49A and -Y49A/F104A). Lower panel shows percentages of Stx16 bound to WT or LDS-mutant GABARAP. Data shown as means ± SEM of precipitated Stx16, n = 3.


WT or STX17 - knockout ( STX17KO ) HeLa cells were starved with or without the presence of bafilomycin A1 ( Baf A1 , 100 nM ) for 2 h , and cell lysates were subjected to Western blot analysis of LC3B and p62 . Quantifications of LC3B - II levels normalized to β - actin from cells treated as in ( A ) ; Data shown as means ± SEM of LC3B - II and β - actin ratios , n = 3 ; * , p < 0 . 05 ( one - way ANOVA ) .

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

WT or STX17-knockout (STX17KO) HeLa cells were starved with or without the presence of bafilomycin A1 (Baf A1, 100 nM) for 2 h, and cell lysates were subjected to Western blot analysis of LC3B and p62.   Quantifications of LC3B-II levels normalized to β-actin from cells treated as in (A); Data shown as means ± SEM of LC3B-II and β-actin ratios, n = 3; *, p < 0.05 (one-way ANOVA).


WT , STX16KO , STX17KO or STX16 / STX17 double KO ( STX16 / STX17DKO ) HeLa cells were starved with or without the presence of Baf A1 ( 100 nM ) for 2 h , and cell lysates were subjected to Western blot analysis of LC3B . Quantifications of LC3B - II levels normalized to β - actin from ( C ) ; Data shown as means ± SEM of LC3B - II and β - actin ratios , n = 3 ; † , not significant ; * * , p < 0 . 01 ( one - way ANOVA ) .

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

WT, STX16KO, STX17KO or STX16/STX17 double KO (STX16/STX17DKO) HeLa cells were starved with or without the presence of Baf A1 (100 nM) for 2 h, and cell lysates were subjected to Western blot analysis of LC3B.   Quantifications of LC3B-II levels normalized to β-actin from (C); Data shown as means ± SEM of LC3B-II and β-actin ratios, n = 3; †, not significant; **, p < 0.01 (one-way ANOVA).


WT or STX16 / STX17DKO HeLa cells were starved in EBSS for 2 h , and subjected to ultrastructural analysis of the autophagic vesicles ( AV ) with electron microscopy . AVi : initial autophagic vacuoles ; AVd : degradative autophagic vacuoles ; G : Golgi apparatus . Image acquisition and counting as in Methods . Scale bars , 1 μm and 0 . 5 µm ( top sections ) . Quantifications of autophagic vesicles in WT and STX16 / STX17DKO HeLa cells treated as ( E ) ; Data shown as means ± SEM of AV profiles relative to cytoplasmic area ; † , not significant ; * , p < 0 . 05 ( two - way ANOVA ) . AV profiles from 47 images of each samples were counted .

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WT or STX16/STX17DKO HeLa cells were starved in EBSS for 2 h, and subjected to ultrastructural analysis of the autophagic vesicles (AV) with electron microscopy. AVi: initial autophagic vacuoles; AVd: degradative autophagic vacuoles; G: Golgi apparatus. Image acquisition and counting as in Methods. Scale bars, 1 μm and 0.5 µm (top sections).   Quantifications of autophagic vesicles in WT and STX16/STX17DKO HeLa cells treated as (E); Data shown as means ± SEM of AV profiles relative to cytoplasmic area; †, not significant; *, p < 0.05 (two-way ANOVA). AV profiles from 47 images of each samples were counted.


WT or STX16 / STX17DKO HeLa - YFP - Parkin cells were treated with CCCP ( 10 µM ) or oligomycin A ( 5 µM ) and antimycin A ( 10 µM ) ( OA ) for 16 hours , and subjected to high - content microscopy ( HCM ) analysis of mitochondria clearance . Masks : blue , nuclei ; red , mitochondria stained with mitochondrial DNA ( mtDNA ) antibody . Scale bar : 20 µm . Quantifications of mtDNA by object count or total area per cell in WT or STX16 / STX17DKO HeLa - YFP - Parkin cells treated as in ( A ) ; Data shown as means ± SEM of mtDNA object count ( upper panel ) or object total area ( lower panel ) per cell ; minimum 500 cells were counted each well from at least 12 wells , 3 independent experiments ; * * , p < 0 . 01 ( two - way ANOVA ) .

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

WT or STX16/STX17DKO HeLa-YFP-Parkin cells were treated with CCCP (10 µM) or oligomycin A (5 µM) and antimycin A (10 µM) (OA) for 16 hours, and subjected to high-content microscopy (HCM) analysis of mitochondria clearance. Masks: blue, nuclei; red, mitochondria stained with mitochondrial DNA (mtDNA) antibody. Scale bar: 20 µm.   Quantifications of mtDNA by object count or total area per cell in WT or STX16/STX17DKO HeLa-YFP-Parkin cells treated as in (A); Data shown as means ± SEM of mtDNA object count (upper panel) or object total area (lower panel) per cell; minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).


WT or STX16 / STX17DKO Huh7 cells were treated with H2O2 ( 0 . 4 mM ) for indicated time points , and autophagic clearance of peroxisomes was measured by the protein levels of PMP70 , PEX14 and p62 . Quantifications of peroxisomal proteins PMP70 and PEX14 for WT or STX16 / STX17DKO Huh7 cells treated as in ( C ) ; Data shown as means ± SEM of PEX14 or PMP70 and β - actin ratios , n = 3 ; † , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ( two - way ANOVA ) .

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

WT or STX16/STX17DKO Huh7 cells were treated with H2O2 (0.4 mM) for indicated time points, and autophagic clearance of peroxisomes was measured by the protein levels of PMP70, PEX14 and p62.   Quantifications of peroxisomal proteins PMP70 and PEX14 for WT or STX16/STX17DKO Huh7 cells treated as in (C); Data shown as means ± SEM of PEX14 or PMP70 and β-actin ratios, n = 3; †, not significant; *, p < 0.05; **, p < 0.01 (two-way ANOVA).


WT , STX16KO , STX17KO or STX16 / STX17DKO THP - 1 cells were infected with Mycobacterium tuberculosis ( Mtb ) , followed by starvation to induce autophagy . Autophagic clearance of infected Mtb was measured by Mtb colonies grown on Middlebrook 7H11 agar plates ; Data shown as means ± SEM of Mtb colonies , n = 4 ; * , p < 0 . 05 ; * * , p < 0 . 01 ( two - way ANOVA ) .

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WT, STX16KO, STX17KO or STX16/STX17DKO THP-1 cells were infected with Mycobacterium tuberculosis (Mtb), followed by starvation to induce autophagy. Autophagic clearance of infected Mtb was measured by Mtb colonies grown on Middlebrook 7H11 agar plates; Data shown as means ± SEM of Mtb colonies, n = 4; *, p < 0.05; **, p < 0.01 (two-way ANOVA).


Validation of STX16 / STX17 DKO by Western blot analysis in HEK293 cells stably expressing RPS3 - Keima ( left panel ) and HCT116 cells stably expressing RPL28 - Keima ( right panel ) .

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Validation of STX16/STX17 DKO by Western blot analysis in HEK293 cells stably expressing RPS3-Keima (left panel) and HCT116 cells stably expressing RPL28-Keima (right panel).


WT or STX16 / STX17DKO HEK293 RPS3 - Keima cells were cultured in full medium or starved for 8 hours , and subjected to HCM analysis of Keima puncta accumulated in autolysosomes with the excitation wavelength at 560 nm . Masks : white , cells identified based on nuclei ; red , Keima puncta detected with the excitation and emission wavelengths of 560 nm and 620 nm , respectively ; lower panel shows Keima puncta without masks . Scale bar : 20 µm . Quantifications of autolysosomal Keima puncta in WT or STX16 / STX17DKO HEK293 RPS3 - Keima cells treated as in ( C ) . Data shown as means ± SEM of Keima puncta per cell , minimum 1000 cells were counted each well from at least 12 wells , 3 independent experiments ; * * , p < 0 . 01 ( two - way ANOVA ) .

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WT or STX16/STX17DKO HEK293 RPS3-Keima cells were cultured in full medium or starved for 8 hours, and subjected to HCM analysis of Keima puncta accumulated in autolysosomes with the excitation wavelength at 560 nm. Masks: white, cells identified based on nuclei; red, Keima puncta detected with the excitation and emission wavelengths of 560 nm and 620 nm, respectively; lower panel shows Keima puncta without masks. Scale bar: 20 µm.   Quantifications of autolysosomal Keima puncta in WT or STX16 /STX17DKO HEK293 RPS3-Keima cells treated as in (C). Data shown as means ± SEM of Keima puncta per cell, minimum 1000 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).


WT or STX16 / STX17DKO HCT116 RPL28 - Keima cells were cultured in full medium or starved for 8 hours , and subjected to HCM analysis of Keima puncta accumulated in autolysosomes with the excitation wavelength at 560 nm . Masks : white , cells identified based on nuclei ; red , Keima puncta detected with the excitation and emission wavelengths of 560 nm and 620 nm , respectively ; lower panel shows Keima puncta without masks . Scale bar : 20 µm . Quantifications of autolysosomal Keima puncta in WT or STX16 / STX17DKO HCT116 RPL28 - Keima cells treated as in ( E ) . Data shown as means ± SEM of Keima puncta per cell , minimum 1000 cells were counted each well from at least 12 wells , 3 independent experiments ; † , not significant ; * * , p < 0 . 01 ( two - way ANOVA ) .

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WT or STX16/STX17DKO HCT116 RPL28-Keima cells were cultured in full medium or starved for 8 hours, and subjected to HCM analysis of Keima puncta accumulated in autolysosomes with the excitation wavelength at 560 nm. Masks: white, cells identified based on nuclei; red, Keima puncta detected with the excitation and emission wavelengths of 560 nm and 620 nm, respectively; lower panel shows Keima puncta without masks. Scale bar: 20 µm.   Quantifications of autolysosomal Keima puncta in WT or STX16/STX17DKO HCT116 RPL28-Keima cells treated as in (E). Data shown as means ± SEM of Keima puncta per cell, minimum 1000 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant; **, p < 0.01 (two-way ANOVA).


( A ) WT or STX16KO HeLa cells were starved in EBSS for 2 h and subjected to HCM analysis of LAMP2 and LC3 puncta . Masks : white , cells identified based on nuclei ; red , LC3 puncta ; green , LAMP2 puncta ; yellow , overlap of LC3 and LAMP2 . Scale bar : 20 µm .

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(A) WT or STX16KO HeLa cells were starved in EBSS for 2 h and subjected to HCM analysis of LAMP2 and LC3 puncta. Masks: white, cells identified based on nuclei; red, LC3 puncta; green, LAMP2 puncta; yellow, overlap of LC3 and LAMP2. Scale bar: 20 µm.


( B - D ) Quantifications of LC3 puncta per cell ( B ) , LC3 and LAMP2 overlap area per cell ( C ) , and LAMP2 puncta per cell ( D ) in WT or STX16KO cells treated Data shown as means ± SEM of puncta or overlap area per cell , minimum 500 cells were counted each well from at least 12 wells , 3 independent experiments ; * , p < 0 . 05 ; * * , p < 0 . 01 ( two - way ANOVA ) .

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(B-D) Quantifications of LC3 puncta per cell (B), LC3 and LAMP2 overlap area per cell (C), and LAMP2 puncta per cell (D) in WT or STX16KO cells treated Data shown as means ± SEM of puncta or overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (two-way ANOVA).


( E ) Western blot analysis of LAMP1 and LAMP2 protein levels in WT and separate clones of STX16KO HeLa , Huh7 , and U2OS cells .

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(E) Western blot analysis of LAMP1 and LAMP2 protein levels in WT and separate clones of STX16KO HeLa, Huh7, and U2OS cells.


( F ) Western blot analysis of LAMP2 protein levels in WT and separate clones of STX6KO or VTI1AKO HeLa cells .

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(F) Western blot analysis of LAMP2 protein levels in WT and separate clones of STX6KO or VTI1AKO HeLa cells.


( G ) Time course of mTOR inactivation during EBSS starvation in WT vs . STX16KO HeLa cells analyzed by Western blotting for mTOR substrates ULK1 and 4E - BP1 .

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(G) Time course of mTOR inactivation during EBSS starvation in WT vs. STX16KO HeLa cells analyzed by Western blotting for mTOR substrates ULK1 and 4E-BP1.


( H ) WT or STX16KO HeLa cells were starved in EBSS for 1 h and subjected to HCM analysis of mTOR puncta . Masks : white , cells identified based on nuclei ; red , mTOR puncta . Scale bar : 20 µm . ( I ) Quantifications of mTOR puncta in WT or STX16KO cells treated as in ( H ) . Data shown as means ± SEM of mTOR puncta per cell , minimum 500 cells were counted each well from at least 12 wells , 3 independent experiments ; * * , p < 0 . 01 ( two - way ANOVA ) .

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(H) WT or STX16KO HeLa cells were starved in EBSS for 1 h and subjected to HCM analysis of mTOR puncta. Masks: white, cells identified based on nuclei; red, mTOR puncta. Scale bar: 20 µm. (I) Quantifications of mTOR puncta in WT or STX16KO cells treated as in (H). Data shown as means ± SEM of mTOR puncta per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).


WT or STX16KO HeLa cells were starved in EBSS for 1 h , followed by starvation with the presence of LysoTracker Red DND - 99 ( LTR ) ( 100 nM ) for additional 30 min , and subjected to HCM analysis of LTR . Scale bar : 20 µm . Quantifications of LTR count per cell or total area per cell in WT and STX16KO HeLa cells treated as in ( A ) . Data shown as means ± SEM of LTR puncta per cell or object total area per cell , minimum 500 cells were counted each well from at least 12 wells , 3 independent experiments ; † , not significant ( two - way ANOVA ) .

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WT or STX16KO HeLa cells were starved in EBSS for 1 h, followed by starvation with the presence of LysoTracker Red DND-99 (LTR) (100 nM) for additional 30 min, and subjected to HCM analysis of LTR. Scale bar: 20 µm.   Quantifications of LTR count per cell or total area per cell in WT and STX16KO HeLa cells treated as in (A). Data shown as means ± SEM of LTR puncta per cell or object total area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant (two-way ANOVA).


WT or STX16KO HeLa cells were starved in EBSS for 1 h , followed by starvation with the presence of LTR ( 100 nM ) for additional 30 min , and subjected to HCM analysis of the colocalization between LTR and TGN46 . Masks : white , cells identified based on nuclei ; red , LTR puncta ; green , TGN46 puncta ; yellow , overlap between LTR and TGN46 . Scale bar : 20 µm . Quantifications of overlaps between LysoTracker Red and TGN46 in WT and STX16KO cells treated as in ( C ) . Data shown as means ± SEM of LTR and TGN46 overlap area per cell , minimum 500 cells were counted each well from at least 12 wells , 3 independent experiments ; * * , p < 0 . 01 ( two - way ANOVA ) .

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WT or STX16KO HeLa cells were starved in EBSS for 1 h, followed by starvation with the presence of LTR (100 nM) for additional 30 min, and subjected to HCM analysis of the colocalization between LTR and TGN46. Masks: white, cells identified based on nuclei; red, LTR puncta; green, TGN46 puncta; yellow, overlap between LTR and TGN46. Scale bar: 20 µm.   Quantifications of overlaps between LysoTracker Red and TGN46 in WT and STX16KO cells treated as in (C). Data shown as means ± SEM of LTR and TGN46 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).


WT or STX16 / STX17DKO HeLa cells were starved in EBSS for 2 h , and subjected to ultrastructural analysis of the Golgi apparatus with electron microscopy . VF of Golgi : volume fraction of Golgi relative to total cytoplasmic area . Scale bar , 2 μm . Red arrows indicate Golgi apparatus .

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

WT or STX16/STX17DKO HeLa cells were starved in EBSS for 2 h, and subjected to ultrastructural analysis of the Golgi apparatus with electron microscopy. VF of Golgi: volume fraction of Golgi relative to total cytoplasmic area. Scale bar, 2 μm. Red arrows indicate Golgi apparatus.


WT , LC3TKO , GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 90 min and subjected to HCM analysis of overlaps between LAMP2 and Stx16 . Masks : white , cells identified based on nuclei ; green , LAMP2 puncta ; red , Stx16 puncta ; yellow , overlap of LAMP2 and Stx16 . Scale bar : 20 µm .

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

WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 90 min and subjected to HCM analysis of overlaps between LAMP2 and Stx16. Masks: white, cells identified based on nuclei; green, LAMP2 puncta; red, Stx16 puncta; yellow, overlap of LAMP2 and Stx16. Scale bar: 20 µm.


Quantifications of overlaps between LAMP2 and Stx16 in WT , LC3TKO , GABARAPTKO or HexaKO HeLa cells treated Data shown as means ± SEM of LAMP2 and Stx16 overlap area per cell , minimum 500 cells were counted each well from at least 12 wells , 3 independent experiments ; * , p < 0 . 05 ; * * , p < 0 . 01 ( one - way ANOVA ) .

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

Quantifications of overlaps between LAMP2 and Stx16 in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells treated Data shown as means ± SEM of LAMP2 and Stx16 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (one-way ANOVA).


Quantifications of overlaps between LBPA and Stx16 in WT , LC3TKO , GABARAPTKO or HexaKO HeLa cells grown in full medium or treated Data shown as means ± SEM of LBPA and Stx16 overlap area per cell , minimum 500 cells were counted each well from at least 12 wells , 3 independent experiments ; * , p < 0 . 05 ; * * , p < 0 . 01 ( one - way ANOVA ) .

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

Quantifications of overlaps between LBPA and Stx16 in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells grown in full medium or treated Data shown as means ± SEM of LBPA and Stx16 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (one-way ANOVA).


WT , LC3TKO , GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 1 hour , followed by starvation with the presence of LTR ( 100 nM ) for additional 30 min , and subjected to HCM analysis of LTR . Scale bar : 20 µm .

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

WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 1 hour, followed by starvation with the presence of LTR (100 nM) for additional 30 min, and subjected to HCM analysis of LTR. Scale bar: 20 µm.


Quantifications of LTR puncta in WT , LC3TKO , GABARAPTKO or HexaKO HeLa cells treated Data shown as means ± SEM of LTR puncta per cell , minimum 500 cells were counted each well from at least 12 wells , 3 independent experiments ; † , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ( one - way ANOVA ) .

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

Quantifications of LTR puncta in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells treated Data shown as means ± SEM of LTR puncta per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant; *, p < 0.05; **, p < 0.01 (one-way ANOVA).


ELISA assay of fecal IgA1 ( A ) , IgA2 ( B ) , total IgA ( C ) , levels from non - IBD ( n = 7 ) , CD ( n = 18 ) , and UC ( n = 12 ) patients ' stool . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney test . P - values are as follows : ( A ) * * : p = 0 , 0098 ; ( B ) * : p = 0 , 0406 ; ( C ) Non - IBD v . UC * : p = 0 , 0479 , CD vs . UC * : p = 0 , 0166 ;

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

ELISA assay of fecal IgA1 (A), IgA2 (B), total IgA (C), levels from non-IBD (n= 7), CD (n=18), and UC (n=12) patients' stool. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (A)** : p= 0,0098; (B) *: p=0,0406; (C) Non-IBD v. UC *: p=0,0479, CD vs. UC *: p=0,0166;


ELISA assay of secretory component ( D ) levels from non - IBD ( n = 7 ) , CD ( n = 18 ) , and UC ( n = 12 ) patients ' stool .

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

ELISA assay of secretory component (D) levels from non-IBD (n= 7), CD (n=18), and UC (n=12) patients' stool.


Data were further separated according to disease activity for IgA1 ( E ) and IgA2 ( F ) . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney test . P - values are as follows : ( F ) * : p = 0 ,

Data were further separated according to disease activity for IgA1 (E) and IgA2 (F). Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (F) *: p=0,0167


In vitro assay of purified IgA1 ( G ) and IgA2 ( H ) reverse - transcytosis abilities on an inverted model of FAE from Caco2 and Raji cells co - culture . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney test . P - values are as follows : ( G ) * : p = 0 , 0441 ; ( H ) * : p = 0 , 423 ; For some patients , antibody purification did not yield a high enough concentration , so samples had to be excluded . N are thus as follows : IgA1 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 4 ; IgA2 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 5 . All patient samples ( biological replicates ) have been tested in technical duplicates meaning nx2 .

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

In vitro assay of purified IgA1 (G) and IgA2 (H) reverse-transcytosis abilities on an inverted model of FAE from Caco2 and Raji cells co-culture. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (G) *: p=0,0441; (H) *: p=0,423 ; For some patients, antibody purification did not yield a high enough concentration, so samples had to be excluded. N are thus as follows: IgA1: Non-IBD: n= 7; CD: n= 8; UC : n=4; IgA2 : Non-IBD: n=7; CD : n= 8; UC: n=5. All patient samples (biological replicates) have been tested in technical duplicates meaning nx2.


( I ) ELISA assay of IgA1 - Dectin - 1 binding , at a rate of one receptor per 10 IgA ; ( J ) ELISA assay of IgA2 - Dectin - 1 binding , at a rate of one receptor per 10 IgA . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney test . P - values are as follows : ( J ) Non - IBD vs . UC * : p = 0 , 0199 , CD vs . UC * : p = 0 , 0243 ; For some patients , antibody purification did not yield a high enough concentration , so samples had to be excluded . N are thus as follows : IgA1 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 4 ; IgA2 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 5 . All patient samples ( biological replicates ) have been tested in technical duplicates meaning nx2 .

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

(I) ELISA assay of IgA1-Dectin-1 binding, at a rate of one receptor per 10 IgA; (J) ELISA assay of IgA2-Dectin-1 binding, at a rate of one receptor per 10 IgA. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (J) Non-IBD vs. UC *: p=0,0199 , CD vs. UC *: p= 0,0243; For some patients, antibody purification did not yield a high enough concentration, so samples had to be excluded. N are thus as follows: IgA1: Non-IBD: n= 7; CD: n= 8; UC : n=4; IgA2 : Non-IBD: n=7; CD : n= 8; UC: n=5. All patient samples (biological replicates) have been tested in technical duplicates meaning nx2.


Percent of IgA1 ( K , L ) and IgA2 ( M , N ) reverse - transcytosis for weak ( K , M ) and strong ( L , N ) Dectin - 1 binding . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney test . P - values are as follows : ( K ) * : p = 0 , 0221 ; ( L ) * : p = 0 , 0486 . For some patients , antibody purification did not yield a high enough concentration , so samples had to be excluded . N are thus as follows : IgA1 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 4 ; IgA2 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 5 . All patient samples ( biological replicates ) have been tested in technical duplicates meaning nx2 .

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

Percent of IgA1 (K, L) and IgA2 (M, N) reverse-transcytosis for weak (K, M) and strong (L, N) Dectin-1 binding. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (K) *: p=0,0221 ; (L) *: p=0,0486 . For some patients, antibody purification did not yield a high enough concentration, so samples had to be excluded. N are thus as follows: IgA1: Non-IBD: n= 7; CD: n= 8; UC : n=4; IgA2 : Non-IBD: n=7; CD : n= 8; UC: n=5. All patient samples (biological replicates) have been tested in technical duplicates meaning nx2.


Heatmaps displaying standardized log2 - transformed signal intensities per glycan for IgA1 ( A ) and IgA2 ( B ) in non - IBD , CD and UC groups , plotted by hierarchical clustering of Euclidean distance .

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

Heatmaps displaying standardized log2-transformed signal intensities per glycan for IgA1 (A) and IgA2 (B) in non-IBD, CD and UC groups, plotted by hierarchical clustering of Euclidean distance.


Flow cytometry analysis of total IgA1 - bound ( A ) and IgA2 - bound ( B ) fecal microbiota . ( C ) Flow cytometry analysis of stool IgA1 + IgA2 - bacteria ( grey dots ) , IgA1 - IgA2 + bacteria ( black dots ) and IgA1 + IgA2 + bacteria ( empty dots ) . ( A - C ) Data were analysed with a Kruskall - Wallis multiple comparison test . In ( C ) * : p = 0 , 0226 . Non - IBD : n = 7 , CD : n = 18 , and UC : n = 12 .

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

Flow cytometry analysis of total IgA1-bound (A) and IgA2-bound (B) fecal microbiota. (C) Flow cytometry analysis of stool IgA1+ IgA2- bacteria (grey dots), IgA1- IgA2+ bacteria (black dots) and IgA1+ IgA2+ bacteria (empty dots). (A-C) Data were analysed with a Kruskall-Wallis multiple comparison test. In (C) *: p= 0,0226. Non-IBD: n= 7, CD: n=18, and UC: n=12.


( 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 .

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

(A-E) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F-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.


( 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 .

(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.


A . 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 .

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

A. 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 .

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

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 .

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

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 .

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

D. 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 .

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

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 .

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

F. 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 .

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

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 .

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

H. 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 .

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

I. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J. 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).


A . 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 .

A. 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 .

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

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 .

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

Representative 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 .

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.


A . 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 .

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

A. 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 .

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

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 .

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

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 .

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 .

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 .

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

F. (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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Immunofluorescence 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.


A . 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 .

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

A. 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 .

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

B. Western blot 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 .

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

C. 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 .

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

D. 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 .

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

E. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Western blot analysis of 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.


A . 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 .

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

A. 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 .

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

B. Western blot 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 ) .

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

C. 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 .

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

D. Quantification 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 .

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

E. 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 .

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

F,G 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 .

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

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 .

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

K. 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.


A . 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 .

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

A. 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 .

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

B. Western blot 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 .

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

C,D. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E,F. 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.


A , 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 .

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

A,B. 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 .

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

C. 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 .

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

D. 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 .

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

E. 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 .

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

F. 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 .

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

G. 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 .

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 .

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I. Representative TEM images of growth plate chondrocytes from mice with indicated genotypes. Arrows indicated the ER. N= nucleus. Scale bar 1μm.


A . 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 .

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

A. 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 .

O O O O O O O O O O O O O O O O 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. 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 .

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

C. 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 .

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.