a , Detection of wild - type ( + ) Ambra1 gene - trap transcripts ( gt ) from total E14 . 5 analysed by northern blotting . marker , 28S ribosomal .

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a, Detection of wild-type (+) and Ambra1 gene-trap transcripts (gt) from total E14.5 embryos analysed by northern blotting. Size marker, 28S ribosomal RNA.


b , Immunoblot of E14 . 5embryobrain extracts using antibodies against Ambra1 ( left panel ) β - gal ( right panel ) . Specific bands ( arrowheads ) unspecific signals ( asterisk ) are indicated . Molecular weights are measured in kilodaltons ( kDa ) . fusion can be revealed only by means of more sensitive anti - β - gal antibody .

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b, Immunoblot analysis of E14.5embryobrain extracts using antibodies against Ambra1 (left panel) and β-gal (right panel). Specific bands (arrowheads) and unspecific signals (asterisk) are indicated. Molecular weights are measured in kilodaltons (kDa). The fusion protein can be revealed only by means of the more sensitive anti-β-gal antibody.


c - f , Expression of Ambra1 in mouse embryonic nervous system . β - gal - on whole - mount Ambra1 + / gt mouseembryos at E8 . 5 ( c ) , E11 . 5 ( d ) on a cross - section of E11 . 5spinal cord ( e ) is shown . Fb , forebrain ; Hb , hindbrain ; Mb , midbrain . f , Matching expression of endogenous gene was revealed by whole - mount Ambra1 mRNA in situ hybridization in E11 . 5 wild - type .

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c-f, Expression of Ambra1 in the mouse embryonic nervous system. β-gal-staining on whole-mount Ambra1+/gt mouseembryos at E8.5 (c), E11.5 (d) and on a cross-section of E11.5spinal cord (e) is shown. Fb, forebrain; Hb, hindbrain; Mb, midbrain. f, Matching expression of the endogenous gene was revealed by whole-mount Ambra1 mRNA in situ hybridization analysis in E11.5 wild-type embryos.


g - l , Electron scanning microscopy of E11 . 5 ( g ) E12 . 5 ( h ) Ambra1gt / gt . Note failure of neural tube closure , extensive midbrain / hindbrain exencephaly ( Ex ) with a closed telencephalon ( T ) , lumbosacral spina bifida ( Sb ) . i , j , E14 . 5 wild - type ( i ) Ambra1gt / gt ( j ) are characterized by prominent exencephaly . k , l , Histological of E12 . 5 wild - type ( k ) Ambra1gt / gt ( l ) on cross - section . Note absence of a normal ventricular system , extensive overgrowth of proliferative neuroepithelium in diencephalon ( Di ) spinal cord ( Sc ) , enlarged fifth ganglia ( VG ) in Ambra1gt / gt .

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g-l, Electron scanning microscopy analysis of E11.5 (g) and E12.5 (h) Ambra1gt/gt embryos. Note the failure of the neural tube closure, the extensive midbrain/hindbrain exencephaly (Ex) with a closed telencephalon (T), and the lumbosacral spina bifida (Sb). i, j, E14.5 wild-type (i) and Ambra1gt/gt (j) embryos are characterized by prominent exencephaly. k, l, Histological analysis of E12.5 wild-type (k) and Ambra1gt/gt (l) embryos on cross-section. Note the absence of a normal ventricular system, the extensive overgrowth of the proliferative neuroepithelium in the diencephalon (Di) and spinal cord (Sc), and the enlarged fifth ganglia (VG) in the Ambra1gt/gt embryo.


m - p , TUNEL ( TdT - mediated dUTP nick end labelling ) of E10 . 5brain ( m , n ) E9 . 5spinal cord ( o , p ) in wild - type ( m , o ) Ambra1gt / gt ( n , p ) sections . q - t , of proliferation in wild - type ( q , s ) Ambra1gt / gt ( r , t ) on transverse sections of E8 . 5cephalic neural folds in prospective hindbrain region ( mitoses , arrows in q , r ) on sagittal sections of E10 . 5forebrain ( BrdU uptake , s , t ) .

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m-p, TUNEL (TdT-mediated dUTP nick end labelling) staining of E10.5brain (m, n) and E9.5spinal cord (o, p) in wild-type (m, o) and Ambra1gt/gt (n, p) embryo sections. q-t, Analysis of cell proliferation in wild-type (q, s) and Ambra1gt/gt (r, t) embryos on transverse sections of E8.5cephalic neural folds in prospective hindbrain region (mitoses, arrows in q, r) and on sagittal sections of E10.5forebrain (BrdU uptake, s, t).


a , AMBRA1 - BECN1 interaction by yeast two - hybrid . Yeast were co - transfected with indicated plasmids plated in medium with ( + ) or without ( - ) histidine ( H ) adenine ( A ) . AD , activation domain ; DBD , - binding domain ; G4 , Gal4 ; LAM , lamin .

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a, AMBRA1-BECN1 interaction by yeast two-hybrid assay. Yeast cells were co-transfected with the indicated plasmids and plated in medium with (+) or without (-) histidine (H) and adenine (A). AD, activation domain; DBD, DNA-binding domain; G4, Gal4; LAM, lamin.


b , AMBRA1 - BECN1 interaction in mammalian . 2FTGH were co - infected with retroviral vectors encoding BECN1 indicated Myc - tagged AMBRA1 , or Myc - tagged β - gal as a negative control . CT , C terminal ; FL , full ; NT , N terminal . extracts were immunoprecipitated using an anti - Myc - tagged antibody ( IP Myc ) . Purified complexes corresponding total extracts were analysed by western blot using anti - Myc ( WB Myc , upper panels ) or anti - BECN1 ( WB BECN1 , lower panels ) antibodies . When multiple bands are present , upper band has expected molecular mass . A scheme of various AMBRA1 mutants is reported .

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b, AMBRA1-BECN1 interaction in mammalian cells. 2FTGH cells were co-infected with retroviral vectors encoding BECN1 and the indicated Myc-tagged AMBRA1 proteins, or Myc-tagged β-gal as a negative control. CT, C terminal; FL, full length; NT, N terminal. Protein extracts were immunoprecipitated using an anti-Myc-tagged antibody (IP Myc). Purified complexes and corresponding total extracts were analysed by western blot using anti-Myc (WB Myc, upper panels) or anti-BECN1 (WB BECN1, lower panels) antibodies. When multiple bands are present, the upper band has the expected molecular mass. A scheme of the various AMBRA1 mutants is reported.


c , AMBRA1 is in a that includes BECN1 Vps34 . extracts from 2FTGH co - expressing BECN1 Myc - tagged AMBRA1 full - ( FL ) or Myc - tagged β - gal were immunoprecipitated using an anti - Myc - tag antibody ( IP Myc ) . Purified complexes corresponding total extracts were analysed using anti - BECN1 ( WB BECN1 ) anti - Vps34 ( WB Vps34 ) antibodies .

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c, AMBRA1 is in a complex that includes BECN1 and Vps34. Protein extracts from 2FTGH cells co-expressing BECN1 and the Myc-tagged AMBRA1 full-length protein (FL) or Myc-tagged β-gal were immunoprecipitated using an anti-Myc-tag antibody (IP Myc). Purified complexes and corresponding total extracts were analysed using anti-BECN1 (WB BECN1) and anti-Vps34 (WB Vps34) antibodies.


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e , BECN1 AMBRA1 co - localize in mammalian . 2FTGH co - expressing BECN1 Myc - tagged AMBRA1 were stained for Myc ( green ) BECN1 ( red ) . Scale bars , 8 µm .

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e, BECN1 and AMBRA1 co-localize in mammalian cells. 2FTGH cells co-expressing BECN1 and Myc-tagged AMBRA1 were stained for Myc (green) and BECN1 (red). Scale bars, 8 µm.


a , AMBRA1 downregulation in 2FTGH using specific short interfering ( siRNA ) oligoribonucleotides ( 1 2 ) . AMBRA1 mRNA were analysed by quantitative polymerase chain reaction ( PCR , left ) western blotting ( right ) , respectively . An unrelated oligoribonucleotide was used as control . Tubulin was used as - loading control . RL , relative . arrowhead indicates AMBRA1 - specific band .

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a, AMBRA1 downregulation in 2FTGH cells using specific short interfering RNA (siRNA) oligoribonucleotides (1 and 2). AMBRA1 mRNA and protein levels were analysed by quantitative polymerase chain reaction (PCR, left) and western blotting (right), respectively. An unrelated oligoribonucleotide was used as the control. Tubulin was used as the protein-loading control. RL, relative levels. The arrowhead indicates the AMBRA1-specific band.


b , Rapamycin - induced autophagy requires AMBRA1 . After AMBRA1 downregulation , 2FTGH were treated with rapamycin ; after 48 h , occurrence of autophagy was analysed by appearance of GFP - LC3 punctate or by LC3 - I to LC3 - II conversion ( western blot in inset ) . Representative results are accompanied by a graph reporting data from three experiments . Scale bar , 20 µm .

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b, Rapamycin-induced autophagy requires AMBRA1. After AMBRA1 downregulation, 2FTGH cells were treated with rapamycin; after 48 h, the occurrence of autophagy was analysed by appearance of GFP-LC3 punctate staining or by LC3-I to LC3-II conversion (western blot in inset). Representative results are accompanied by a graph reporting data from three experiments. Scale bar, 20 µm.


c , Nutrient - starvation - induced autophagy requires AMBRA1 . After AMBRA1 downregulation , 2FTGH were starved for 4 h analysed for appearance of GFP - LC3 punctate .

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c, Nutrient-starvation-induced autophagy requires AMBRA1. After AMBRA1 downregulation, 2FTGH cells were starved for 4 h and analysed for appearance of GFP-LC3 punctate staining.


d , AMBRA1 overexpression increases basal rapamycin - induced autophagy . 2FTGH were transduced with full - AMBRA1 ( FL ) , AMBRA1 fragments ( F1 - 3 ) , BECN1 β - gal ( negative control ) encoding retroviruses . They were then stimulated with rapamycin or left untreated , analysed by appearance of GFP - LC3 punctate .

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d, AMBRA1 overexpression increases basal and rapamycin-induced autophagy. 2FTGH cells were transduced with full-length AMBRA1 (FL), AMBRA1 fragments (F1-3), BECN1 and β-gal (negative control) encoding retroviruses. They were then stimulated with rapamycin or left untreated, and analysed by appearance of GFP-LC3 punctate staining.


e , BECN1 - induced autophagy requires AMBRA1 . After AMBRA1 downregulation , GFP - LC3 - expressing 2FTGH were transduced with a BECN1 - encoding retrovirus . Occurrence of autophagy was analysed 48 h later by appearance of GFP - LC3 punctate .

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e, BECN1-induced autophagy requires AMBRA1. After AMBRA1 downregulation, GFP-LC3-expressing 2FTGH cells were transduced with a BECN1-encoding retrovirus. Occurrence of autophagy was analysed 48 h later by appearance of GFP-LC3 punctate staining.


f , AMBRA1 downregulation reduces amount of Vps34 associated to BECN1 during autophagy . Forty - eight hours after AMBRA1 downregulation with oligo 2 , 2FTGH were either starved or cultured in standard medium for an additional 4 h . extracts were immunoprecipitated ( IP ) using an anti - BECN1 antibody . Purified complexes corresponding total extracts were analysed by western blotting with anti - Vps34 ( left panels ) anti - BECN1 ( right panels ) antibodies . graph indicates signal of BECN1 - associated Vps34 as determined by densitometry .

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f, AMBRA1 downregulation reduces the amount of Vps34 associated to BECN1 during autophagy. Forty-eight hours after AMBRA1 downregulation with oligo 2, 2FTGH cells were either starved or cultured in standard medium for an additional 4 h. Protein extracts were immunoprecipitated (IP) using an anti-BECN1 antibody. Purified complexes and corresponding total extracts were analysed by western blotting with anti-Vps34 (left panels) and anti-BECN1 (right panels) antibodies. The graph indicates the signal intensity of BECN1-associated Vps34 as determined by densitometry.


a , Alteration of both LC3 conversion translocation in Ambra1gt / gt . Left panel : immunoblot of LC3 in E14 . 5embryos . LC3 - I to LC3 - II conversion is reduced in mutant extracts ( LC3 - II / LC3 - I , as measured by densitometry , from left to right : 5 % , 9 . 8 % , 2 . 5 % , 4 . 6 % ) . B , ; H , head . Centre right panels : sections from E10 . 5Ambra1 + / + ; GFP - LC3 Ambra1gt / gt ; GFP - LC3neuroepithelia were compared to evaluate GFP - LC3 subcellular distribution . Clusters of GFP - LC3 punctate structures ( arrows ) are indicated in wild - type sections . were optimized by deconvolution . Scale bar , 10 µm .

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a, Alteration of both LC3 conversion and translocation in Ambra1gt/gt embryos. Left panel: immunoblot analysis of LC3 in E14.5embryos. LC3-I to LC3-II conversion is reduced in mutant extracts (LC3-II/LC3-I ratio, as measured by densitometry, from left to right: 5%, 9.8%, 2.5%, 4.6%). B, body; H, head. Centre and right panels: sections from E10.5Ambra1+/+;GFP-LC3 and Ambra1gt/gt;GFP-LC3neuroepithelia were compared to evaluate GFP-LC3 subcellular distribution. Clusters of GFP-LC3 punctate structures (arrows) are indicated in wild-type sections. Images were optimized by deconvolution. Scale bar, 10 µm.


c , Rapamycin - induced autophagy is impaired in Ambra1gt / gt murine embryonic fibroblasts ( MEFs ) . dissected from with different genotypes ( A - D ) were treated with rapamycin , occurrence of autophagy was analysed 48 h later by appearance of GFP - LC3 punctate . Representative results are shown in upper panels , whereas lower panel shows data from three experiments . Scale bar , 20 µm .

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c, Rapamycin-induced autophagy is impaired in Ambra1gt/gt murine embryonic fibroblasts (MEFs). Cells dissected from embryos with different genotypes (A-D) were treated with rapamycin, and the occurrence of autophagy was analysed 48 h later by the appearance of GFP-LC3 punctate staining. Representative results are shown in the upper panels, whereas the lower panel shows the data from three experiments. Scale bar, 20 µm.


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Example showing localization of SOX2 - SNAP OCT4 - HALO by microscopy of SBROS . Scale bar : 50µm .

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Example images showing the localization of SOX2-SNAP and OCT4-HALO by fluorescence microscopy of SBROS cells. Scale bar: 50µm.


Correlation between OCT4 - HALO total OCT4 determined by immunofluorescence HALO labelling . R is Pearson ' s correlation coefficient .

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Correlation between OCT4-HALO and total OCT4 levels determined by immunofluorescence and HALO labelling. R is Pearson's correlation coefficient.


Distributions of OCT4 ( n = 2682 ) , SOX2 ( n = 1236 ) NANOG ( n = 2416 ) in WT E14 SBROS lines as determined by quantitative immunofluorescence . Dashed lines : mean ; Dotted lines : median .

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Distributions of OCT4 (n=2682), SOX2 (n=1236) and NANOG (n=2416) levels in WT E14 and SBROS cell lines as determined by quantitative immunofluorescence. Dashed lines: mean protein levels; Dotted lines: median protein levels.


half - lives of OCT4 - HALO SOX2 - SNAP in SBROS . Whiskers : minimum maximum values ; Box : lower upper quartiles ; Solid : median . Black dots : outliers .

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Protein half-lives of OCT4-HALO and SOX2-SNAP in SBROS cells. Whiskers: minimum and maximum values; Box: lower and upper quartiles; Solid line: median. Black dots: outliers.


cycle duration of E14 WT ( n = 6 ) SBROS ( n = 8 ) . Error bars : SE . p - value : two - sided t - with unequal variance .

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Cell cycle duration of E14 WT (n=6) and SBROS (n=8) cells. Error bars: SE. p-value: two-sided t-test with unequal variance.


Endogenous SOX2 in single ( turquoise ) normalized to value at t = 0 in SNSF upon induction of SOX2 - SNAP ( n = 37 , left ) or a truncated SOX2 version missing binding domain ( YPET - SOX2 - delDBD , n = 47 , right ) . Black : average SOX2 - SNAP after induction normalized to maximum level ( n = 22 ) ; Yellow : average YPET - SOX2 - delDBD after induction , normalized to maximum level ( n = 20 ) .

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Endogenous SOX2 levels in single cells (turquoise) normalized to the value at t=0 in SNSF cells upon induction of SOX2-SNAP (n=37, left) or a truncated SOX2 version missing the DNA binding domain (YPET-SOX2-delDBD, n=47, right). Black line: average SOX2-SNAP levels after induction normalized to the maximum level (n=22); Yellow line: average YPET-SOX2-delDBD levels after induction, normalized to the maximum level (n=20).


Sox2 Oct4 mRNA upon overexpression of YPet - SOX2 or YPET - SOX2 - delDBD ( n = 3 ) . p - values : two - sided t - with unequal variance .

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Sox2 and Oct4 mRNA levels upon overexpression of YPet-SOX2 or YPET-SOX2-delDBD (n=3). p-values: two-sided t-test with unequal variance.


Changes of OCT4 half - life upon SOX2 - SNAP or YPET - SOX2 - delDBD overexpression ( n = 20 each ) . p - values : Mann - Whitney U . SBROS harbouring inducible SOX2 - SNAP or YPET - SOX2 - delDBD were treated with 0 or 500 ng / ml dox for 6 - 8 hours followed by - labelling of OCT4 - HALO live - imaging .

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Changes of OCT4 half-life upon SOX2-SNAP or YPET-SOX2-delDBD overexpression (n=20 cells each). p-values: Mann-Whitney U test. SBROS cells harbouring inducible SOX2-SNAP or YPET-SOX2-delDBD were treated with 0 or 500 ng/ml dox for 6-8 hours followed by pulse-labelling of OCT4-HALO and live-cell imaging.


Sorting strategy to evaluate impact of endogenous fluctuations of SOX2 OCT4 on OCT4 SOX2 , respectively .

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Sorting strategy to evaluate the impact of endogenous fluctuations of SOX2 and OCT4 on OCT4 and SOX2 protein levels, respectively.


Changes of SOX2 OCT4 after sorting for high low SOX2 in expressing intermediate OCT4 ( n = 4 ) . Error bars : SE ; p - values : one - sided t - with unequal variance .

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Changes of SOX2 and OCT4 levels after sorting for high and low SOX2 levels in cells expressing intermediate OCT4 levels (n=4). Error bars: SE; p-values: one-sided t-test with unequal variance.


Changes of SOX2 OCT4 after sorting for high low OCT4 in expressing intermediate SOX2 ( n = 4 ) . p - values : one - sided t - with unequal variance .

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Changes of SOX2 and OCT4 levels after sorting for high and low OCT4 levels in cells expressing intermediate SOX2 levels (n=4). p-values: one-sided t-test with unequal variance.


Representative traces of absolute number of SOX2 - NLUC molecules ( top ) inferred in nM ( bottom ) in in - silico synchronized ( n = 10 ) . Single were ranked according to their SOX2 expression at t = 0 assigned a color code for their initial rank from low ( blue ) to high ( red ) , changes in ranks over time are shown ( n = 59 ) . Rank - autocorrelation function of SOX2 ranks ( n = 59 ) . Error bars : SE estimated by bootstrapping .

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Representative traces of the absolute number of SOX2-NLUC molecules (top) and inferred concentration in nM (bottom) in in-silico synchronized cells (n=10). Single cells were ranked according to their SOX2 expression at t=0 and assigned a color code for their initial rank from low (blue) to high (red) levels, and changes in ranks over time are shown (n=59). Rank-based autocorrelation function of the SOX2 ranks (n=59). Error bars: SE estimated by bootstrapping.


Representative single traces of integrated of OCT4 - HALO in single ( top ) corresponding inferred ( bottom ) in in - silico synchronized ( n = 10 ) . Representation of ranks as in G . Rank - autocorrelation function as in I ( n = 47 ) . Error bars : SE estimated by bootstrapping .

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Representative single cell traces of the integrated intensity of OCT4-HALO in single cells (top) and the corresponding inferred concentration (bottom) in in-silico synchronized cells (n=10). Representation of the ranks as in G. Rank-based autocorrelation function as in I (n=47). Error bars: SE estimated by bootstrapping.


Snapshot from luminescence movie of SNSF in differentiation . arrowheads : starting to express SOX1 - P2A - FLUC ; Orange arrowheads : staying SOX1 - negative throughout experiment . Red : SOX2 - NLUC , turquoise : Sox1 - P2A - FLUC . Scale bar : 50µm .

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Snapshot from luminescence movie of SNSF cells in differentiation. White arrowheads: cells starting to express SOX1-P2A-FLUC; Orange arrowheads: cells staying SOX1-negative throughout the experiment. Red: SOX2-NLUC, turquoise: Sox1-P2A-FLUC. Scale bar: 50µm.


Single traces of SOX2 in differentiating . Turquoise traces : becoming SOX1 - positive in middle cycle , population average is shown in bold black ; Red traces : remaining SOX1 - negative throughout experiment , population average is shown in bold ; Green : population average FLUC signal in that become SOX1 - positive . * p < 0 . 05 determined by two - sided t - with unequal variance .

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Single cell traces of SOX2 levels in differentiating cells. Turquoise traces: cells becoming SOX1-positive in the middle cell cycle, population average is shown in bold black; Red traces: cells remaining SOX1-negative throughout the experiment, population average is shown in bold; Green line: population average FLUC signal in cells that become SOX1-positive. * p<0.05 determined by two-sided t-test with unequal variance.


Strategies to sort SBROS populations with different SOX2 OCT4 . Differentiation outcome for sorted for different SOX2 ( n = 5 for low high , n = 4 for medium ) . p - values : two - sided t - with unequal variance . Differentiation outcome for sorted for different OCT4 ( n = 4 ) . p - values : two - sided t - with unequal variance .

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Strategies to sort SBROS cell populations with different SOX2 and OCT4 levels. Differentiation outcome for cells sorted for different SOX2 levels (n=5 for low and high, n=4 for medium). p-values: two-sided t-test with unequal variance. Differentiation outcome for cells sorted for different OCT4 levels (n=4). p-values: two-sided t-test with unequal variance.


Differentiation outcome of uninduced or induced 12 hours for OCT4 - SNAP overexpression before sorting in G1 phase ( n = 6 ) . p - values : two - sided t - with unequal variance .

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Differentiation outcome of uninduced cells or cells induced 12 hours for OCT4-SNAP overexpression before sorting in G1 phase (n=6). p-values: two-sided t-test with unequal variance.


Sorting strategy for SBROS in G1 or S phase into four following subpopulations : SOX2 high & OCT4 high ( SHOH ) , SOX2 high & OCT4 low ( SHOL ) , SOX2 low & OCT4 high ( SLOH ) or SOX2 low & OCT4 low ( SLOL ) .

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Sorting strategy for SBROS cells in G1 or S phase and into the four following subpopulations: SOX2 high & OCT4 high (SHOH), SOX2 high & OCT4 low (SHOL), SOX2 low & OCT4 high (SLOH) or SOX2 low & OCT4 low (SLOL).


Differentiation outcome for sorted in G1 phase ( n = 5 ) . p - values : two - sided t - with unequal variance ( SOX1 ) Mann - Whitney U ( BRA ) . Differentiation outcome for sorted in S phase ( n = 5 ) . p - values : Mann - Whitney U ( SOX1 ) two - sided t - with unequal variance ( BRA ) .

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Differentiation outcome for cells sorted in G1 phase (n=5). p-values: two-sided t-test with unequal variance (SOX1) and Mann-Whitney U test (BRA). Differentiation outcome for cells sorted in S phase (n=5). p-values: Mann-Whitney U test (SOX1) and two-sided t-test with unequal variance (BRA).


Differentiation outcome for sorted in G1 phase differentiated in presence of 3μM CHIR ( n = 7 ) . p - values : two - sided t - with unequal variance .

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Differentiation outcome for cells sorted in G1 phase differentiated in the presence of 3μM CHIR (n=7). p-values: two-sided t-test with unequal variance.


Differentiation outcome for sorted in G1 phase differentiated in presence of 1 μM SB431542 25 ng / ml bFGF ( n = 7 ) . p - values : two - sided t - with unequal variance .

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Differentiation outcome for cells sorted in G1 phase differentiated in the presence of 1 μM SB431542 and 25 ng/ml bFGF (n=7). p-values: two-sided t-test with unequal variance.


Example tracks of ATAC - seq signal in SHOH , SHOL , SLOH SLOL populations for loci where accessibility is unaffected , affected by OCT4 , SOX2 , or affected by both .

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Example tracks of the ATAC-seq signal in SHOH, SHOL, SLOH and SLOL populations for loci where chromatin accessibility is unaffected, affected by OCT4 levels, SOX2 levels, or affected by both.


of relative accessibility in SHOH / SHOL / SLOH / SLOL samples for loci with significantly altered accessibility , with examples of genes close to affected regulatory regions . Groups are defined by their significance ( OHvsOL / SHvsSL / SHOHvsSLOL ) fold - change direction .

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Heatmap of relative chromatin accessibility in SHOH/SHOL/SLOH/SLOL samples for all loci with significantly altered accessibility, with examples of genes close to affected regulatory regions. Groups are defined by their significance (OHvsOL/SHvsSL/SHOHvsSLOL) and fold-change direction.


of regions in each group showing an overlap with OCT4 , SOX2 H3K4me3 ChIP - seq peaks .

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Percentage of regions in each group showing an overlap with OCT4, SOX2 and H3K4me3 ChIP-seq peaks.


A . MS - proteomic of RCS chondrocytes treated with vehicle ( 5 % ABS ) FGF18 ( 50 ng / mL ) for 16 hours . Biological processes 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 on Wilcoxon - Mann - Whitney .

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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 of Lysotracker ( B ) dye in RCS treated with indicated FGF ligands ( 50 ng / mL ) for 16h . intensities were expressed as % relative to vehicle ( 5 % ABS ) . Mean + / - standard error of mean ( ) of N = 3 biological replicates / treatment . of variance one way ( ANOVA ) p < 0 . 002 ( B ) Tukey ' s post hoc * * * p < 0 . 0005 .

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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 of DQ - BSA ( C ) dye in RCS treated with indicated FGF ligands ( 50 ng / mL ) for 16h . Concanamycin A was used at 100 nM for 1h to inhibit lysosomal function . intensities were expressed as % relative to vehicle ( 5 % ABS ) . Mean + / - standard error of mean ( ) of N = 3 biological replicates / treatment . of variance one way ( ANOVA ) = 0 . 005 ( C ) ; Tukey ' s post hoc * * * p < 0 . 0005 .

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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 ) FGF18 ( 50 ng / mL ) for 16 hours . Insets show magnification of boxed . Nuclei were stained with DAPI ( blue ) . Scale bar 15 μm 2 μm ( higher magnification boxes ) . Bar graph shows of Lamp1 - positive vesicles / . Mean + / - standard error of mean ( ) of N = 3 biological replicates / treatment . n = 45 were analyzed . Student paired T - * p < 0 . 05 .

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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 of RCS chondrocytes treated with 5 % ABS ( vehicle ) 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 . shows average number of ER membranes mitochondria number / lysosome vesicle ( Lys ) . Mean + / - standard error of mean ( ) of N = 3 biological replicates / treatment . ER fragment / vesicle n = 60 ( vehicle ) n = 72 ( FGF18 ) were analyzed ; Mitochondria number / vesicle : n = 40 ( vehicle ) n = 72 ( FGF18 ) were analyzed . Student paired T - * * * p < 0 . 0005 ; NS not significant .

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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 of ER ( ER tracker BODIPY green ) lysosomes ( Lamp1 , red ) in RCS chondrocytes treated with vehicle ( 5 % ABS ) FGF18 ( 50 ng / mL ) for 16 hours . Insets show higher magnification of boxed . Scale bar 15 μm 2 μm ( higher magnification boxes ) . N = 3 biological replicates / treatment .

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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 : eGFP , but not mCherry , is lost at acidic pH . Chondrocytes with indicated genotypes ( ctrl = wild type ) were treated with FGF18 ( 50 ng / mL ; 12 hours ) BafA1 ( 200 nM ; 3 hours ) where indicated . ER acidification was measured by FACS . Mean + / - standard error of mean ( ) of N = 3 biological replicates . of variance one way ( ANOVA ) p < 0 . 0001 ; Tukey ' s post hoc * * * p < 0 . 0005 ; * * p < 0 . 005 ; NS not significant .

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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 of DQ - BSA dye 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 . intensities were expressed as % relative to vehicle ( 5 % ABS ) . Mean + / - standard error of mean ( ) of N = 3 biological replicates / treatment / genotype . of variance ( ANOVA ) p = 0 . 00279 ; Tukey ' s post hoc * * p < 0 . 005 ; * p < 0 . 05 ; NS not significant .

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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 . 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 mean ( ) of N = 17 ( veh ) , N = 17 ( FGF18 ) , N = 4 ( FGFR3 ; 4KO ) ; N = 4 ( FGFR3 ; 4KO FGF18 ) biological replicates . of variance one way ( ANOVA ) p < 0 . 0001 ; Tukey ' s post hoc * * * p < 0 . 0005 ; NS not significant .

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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 of ER ( CLIMP63 , green ) 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 . of relative CLIMP63 into TMEM192 - HA positive vesicles . N = 3 biological replicates ; n = 21 were analyzed . Mean + / - standard error of mean ( ) . Paired Student ' s T - NS = not significant . Scale bar 10 μm 1 . 3 μm ( higher magnification boxes ) .

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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 of RCS with indicated genotypes ( ctrl = wild type ) treated with vehicle ( 5 % ABS ) 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 .

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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 of IRS1 , p - P70S6K ( T389 ) , P70S6K , p - AKT ( T308 ) , AKT in RCS chondrocytes treated with vehicle ( 5 % ABS ) 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 of indicated normalized to their totals . IRS1 was normalized to β - actin . Mean + / - standard error of mean ( ) . Student ' s paired T - * * p < 0 . 005 ; * p < 0 . 05 ; NS not significant .

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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 of IRS1 , p - P70S6K ( T389 ) , P70S6K , p - AKT ( T308 ) , AKT , p - cJUN ( s73 ) cJUN in RCS chondrocytes treated with vehicle ( 5 % ABS ) 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 of indicated normalized to their totals . IRS1 was normalized to β - actin . Mean + / - standard error of mean ( ) . Student ' s paired T - * * p < 0 . 005 ; * p < 0 . 05 ; NS not significant .

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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 ) p - S6 S240 / S242 ( red ) ribosomal in IRS1 - overexpressing RCS chondrocytes treated with FGF18 ( 50 ng / mL ) for 12h . Nuclei ( N ) were stained with DAPI ( blue ) . Scale bar 15 μm . of p - S6 in IRS1 - overexpressing vs non expressing RCS chondrocytes . Mean + / - standard error of mean ( ) of N = 3 biological replicates . n = 35 analyzed . Student ' s paired T - * p < 0 . 05 .

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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 . Chondrocytes were treated with FGF18 ( 50 ng / mL ; 16 hours ) Actinomycin ( 1μg / mL ; last 4 hours ) where indicated . ER acidification was measured by FACS . Mean + / - standard error of mean ( ) of N = 17 ( veh ) , N = 17 ( FGF18 ) , N = 5 ( actinomycin ) , N = 5 ( actinomycin + FGF18 ) biological replicates . of variance one way ( ANOVA ) p < 0 . 0001 ; Tukey ' s post hoc * * * p < 0 . 0005 ; NS not significant .

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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 of Lysotracker dye in RCS chondrocytes treated with FGF18 ( 50 ng / mL ) for 16 hours . Actinomycin ( 1μg / mL ) was added for last 4 hours . intensities were calculated as % relative to vehicle ( 5 % ABS ) . Mean + / - standard error of mean ( ) of N = 3 biological replicates . of variance one way ( ANOVA ) p = 0 . 0002 ; Tukey ' s post hoc * * * p < 0 . 0005 ; NS not significant .

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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 of Lamp1 ( red ) in RCS chondrocytes treated with vehicle ( 5 % ABS ) , FGF18 ( 50 ng / mL ; 16 hours ) Actinomycin ( 1μg / mL ; 4 hours ) . Nuclei were stained with DAPI ( blue ) . Scale bar 10 μm . Representative of N = 3 biological replicates . Scale bar 10 μm .

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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 autophagy genes expression in RCS chondrocytes with indicated genotypes ( ctrl = wild type ) , treated with vehicle ( 5 % ABS ) 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 .

O O O O O O O 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

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 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 ) normalized to Cyclophilin gene . Mean + / - standard error of mean ( ) of N = 3 biological replicates / treatment . Student ' s paired T - * * 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 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

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 . LIR = LC3 - Interacting - Region . ( bottom ) Western blot of Fam134b in chondrocytes with indicated genotypes ( ctrl = wild type ) treated with FGF18 ( 50 ng / mL ) for 16 hours . Representative of N = 3 biological replicates / treatment . β - actin was used as loading control . Bar graph showed of Fam134b band normalized to β - actin . Mean + / - standard error of mean ( ) . Student ' s paired T - , * p < 0 . 05 .

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

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 of CLIMP63 ( green ) 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 . of CLIMP63 into TMEM192 - HA decorated lysosomes . Mean + / - standard error of mean ( ) of N = 3 biological replicates . n = 10 / experiment were analyzed . of variance one way ( ANOVA ) p = 1 . 55e - 7 ; Tukey ' s post hoc * * * p < 0 . 0005 ; NS not significant . H . EATR in RCS with indicated genotypes ( ctrl = wild type ) showing % of with acidified ER by FACS . FGF18 was used at 50 ng / mL for 16 hours . Mean + / - standard error of mean ( ) of N = 4 biological replicates / treatment / genotype . of variance one way ( ANOVA ) p < 0 . 0001 ; Tukey ' s post hoc * * * p < 0 . 0005 ; NS not significant .

O O O O B-MEASURED_VAR 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 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 B-MEASURED_VAR 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 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

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 ( expressed as % ) of with TFE3 TFEB ( representative of TFEB immunofluorescence are shown in EV2E ) . Mean + / - standard error of mean ( ) of N = 3 biological replicates ; n = 80 ( TFE3 ) n = 70 ( TFEB ) / experiment were analyzed . Scale bar 10 μm . of variance one - way ( ANOVA ) p = 3 . 23e - 12 ( TFEB ) , p = 2 . 48e - 11 ( TFE3 ) ; Tukey ' s post hoc * * * p < 0 . 0005 ; NS not significant .

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

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 of TFEB , phospo - TFEB ( Serine 142 ) in RCS chondrocytes stably expressing human TFEB - 3XFlag . 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 B-MEASURED_VAR 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

B. 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 in FGFR3 / 4KO chondrocytes infected with a constitutive ( active ) mutant TFEB fused to GFP tag ( TFEB - S142A : S211A - GFP ) . GFP positive gold immune - particles showed presence of GFP - into nucleus ( N , stained in green for visualization ) . Lysosomes were stained in blue for visualization . Insets showed magnification of lysosomes . of lysosome diameter ( nm ) in control ( wild type ) FGFR3 ; 4KO chondrocytes infected with empty or with TFEB - S142A : S211A - GFP vector . Mean + / - standard error of mean ( ) of N = 3 biological replicates . n = 40 ( veh ) n = 78 ( FGFR3 ; 4KO ) n = 57 ( FGFR3 ; 4KO + TFEB - S142A : S211A - GFP ) were analyzed . Scale bar 500 nm . Kruskal Wallis p = 1 . 43e - 13 ; Nemenyi post hoc * * * p < 0 . 0005 ; NS not significant .

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

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 ) TFEB in IRS1 - overexpressing RCS chondrocytes treated with FGF18 ( 50 ng / mL ) for 12h . Nuclei ( N ) were stained with DAPI ( blue ) delimited with dashed . Scale bar 15 μm . of TFEB localization in IRS1 - overexpressing vs non - expressing RCS . Mean + / - standard error of mean ( ) of N = 3 biological replicates . n = 124 analyzed ; Student ' s un - paired T - * p < 0 . 05 .

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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 of TFEB ( red ) 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 ) . showed % of with TFEB TFE3 in RCS chondrocytes with indicated treatments . Scale bar 15 μm . Mean + / - standard error of mean ( ) of N = 3 biological replicates . n = 126 ( control ) , 126 ( FGF18 ) , 95 ( JNK Inhibitor ) , 163 ( JNK Inhibitor + FGF18 ) . Student ' s paired T - * p < 0 . 05 .

O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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 of phospho - TFEB ( S142 ) IRS1 in RCS chondrocytes treated with vehicle ( 5 % ABS ) 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 of N = 3 independent experiments . Bar graphs show of indicated normalized to β - actin . Mean + / - standard error of mean ( ) . Student ' s paired T - * p < 0 . 05 ; NS not significant .

O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 normalized to Cyclophilin gene . Mean + / - standard error of mean ( ) of N = 3 biological replicates . of variance one way ( ANOVA ) p < 0 . 0001 : Tukey ' s post hoc * * * p < 0 . 0005 ; NS not significant .

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

A. 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 of Fam134b in chondrocytes with indicated genotypes treated with vehicle ( 5 % ABS ) FGF18 ( 50 ng / mL ) overnight . β actin was used as loading control . Representative image of N = 3 biological replicates . Bar graph showed of Fam134b normalized to β - actin . of variance one way ( ANOVA ) p < 0 . 0001 ; Sidak ' s multiple comparison * * * p < 0 . 0005 ; * * p < 0 . 005 NS not significant .

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

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 - of CLIMP63 ( green ) TMEM192 - HA ( lysosomes , red ) in control TFEB ; 3KO RCS treated with FGF18 ( 50 ng / mL for 16 hours ) . BaFA1 was used at 100 nM for 3h . Scale bar 15 μm 2 μm ( higher magnification boxes ) .

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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 . of CLIMP63 into TMEM192 - HA decorated lysosomes . Mean + / - standard error of mean ( ) of N = 3 biological replicates / treatment / genotype ( ctrl = wild type ) . n = 10 / experiment were analyzed . of variance one way ( ANOVA ) p = 1 . 55e - 7 ; Tukey ' s post hoc * * * p < 0 . 0005 ; NS not significant .

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

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 in chondrocytes with indicated genotypes ( ctrl = wild type ) showing % of with acidified ER measured by FACS . FGF18 was used at 50 ng / mL overnight . Mean + / - standard error of mean ( ) of N = 4 biological replicates . of variance one way ( ANOVA ) p < 0 . 0001 : Tukey ' s post hoc * * * p < 0 . 0005 ; * p < 0 . 05 ; NS not significant .

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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 of mCherry + vesicles / ( autolysosomes ) ( F ) mCherry + / GFP + vesicles / ( autophagosomes ) ( G ) in wild type ( ctrl ) TFEB ; 3KO treated with vehicle ( veh ) or FGF18 . N = 3 independent experiments . Mean + / - standard error of mean ( ) 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 ) . Student un - paired T - * * * 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 B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR 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

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 of TFEB binding to Fam134b in RCS transfected with TFEB - 3XFLAG . Numbers in CLEAR site ( yellow box ) refer to [ in base pairs ] from transcriptional start site ( + 1 ) of Fam134b - 2 gene . Immunoprecipitated was normalized to input plotted as relative enrichment over a mock control . Bar graph shows fold change enrichment ; mean + / - standard error of mean ( ) of N = 3 independent experiments . Student un - paired T - * * p < 0 . 005 .

O O O O B-MEASURED_VAR 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 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 fragment containing a wild type ( FAM134B - WT ) or a deleted ( FAM134B - mut ) version of CLEAR site . TFEB plasmid transfection amount FGF18 ( 50 ng / mL for 16h ) treatments are indicated . Mean + / - standard error of mean ( ) of N = 3 biological replicates . Student paired T - * p < 0 . 05 ; * * p < 0 . 005 .

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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 ) TFEB ; 3KO RCS chondrocytes with indicated genotypes expressing indicated Fam134b luciferase report plasmids treated with FGF18 overnight ( 50 ng / mL ) where indicated . Mean + / - standard error of mean ( ) of N = 5 biological replicates . of variance one way ( ANOVA ) p < 0 . 0001 : Sidak ' s multiple comparison * * * p < 0 . 0005 ; NS not significant .

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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 of Fam134b isoforms in mock , TFEB S142A : S211A TFE3 S246A : S312A over - expressing U2OS . Values were normalized to Hprt gene expressed as fold change values relative to mock . Mean + / - standard error of mean ( ) of N = 3 biological replicates . Student ' s un - paired T - * p < 0 . 05 ; NS not significant .

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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 of Fam134b isoforms in mock , TFEB S142A : S211A TFE3 S246A : S312A over - expressing U2OS 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 .

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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 of ER ( CLIMP - 63 , green ) lysosomes ( TMEM192 - HA , red ) in control Sh - FAM134B U2OS over - expressing TFEB S142A : S211A - GFP ( purple ) . BaFA1 was used at 100 nM for 4h . Scale bar 15 μm 2 μm ( higher magnification boxes ) ( C ) . In ( D ) , of CLIMP63 in TMEM192 - HA decorated lysosomes . Mean + / - standard error of mean ( ) 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 ) were analysed . Student ' s un - paired T - * * * p < 0 . 0005 ; NS not significant .

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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 of ER ( CLIMP - 63 , green ) lysosomes ( Lamp1 , red ) in WT Fam134bKO MEF over - expressing TFEB S142A : S211A - GFP ( purple ) . BaFA1 was used at 100 nM for 4h . Scale bar 15 μm 2 μm ( higher magnification boxes ) ( E ) . In ( F ) , of CLIMP63 in Lamp1 decorated lysosomes . Mean + / - standard error of mean ( ) 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 ) were analysed . Student ' s un - paired T - * * * p < 0 . 0005 ; NS not significant .

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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 of alcian blue ( cartilage ) Alizarin red ( bone ) skeletal showing growth retardation in Fgfr3 / 4 dKO mice compared to / sex wild type littermate at post - natal day 30 . B . Femur , tibia tail details .

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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 of femoral growth plate sections from wild type Fgfr3 / 4 dKO mice . Higher magnification insets showed a disorganized hypertrophic chondrocyte layer , in Fgfr3 / 4 dKO mice . Scale bar 60 μm .

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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 of IRS1 , p - P70S6K ( T389 ) , P70S6K , p - JNK ( Y185 ) , JNK1 / 2 in growth plate lysates of mice with indicated genotypes . N = 3 mice / genotype . β - actin was used as loading control . Bar graph shows . Mean + / - standard error of mean ( ) . Student ' s un - paired T - * p < 0 . 05 .

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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 of LC3 Lamp1 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 . Mean + / - standard error of mean ( ) . Student ' s un - paired T - * p < 0 . 05 .

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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 of Fam134b - 2 expression from growth plate of mice with indicated genotypes . N = 8 ( wt mice ) N = 9 ( Fgfr3 / 4 dKO mice ) were analyzed . Values were normalized to Hprt gene expressed as fold change relative to control . Mean + / - standard error of mean ( ) . Student ' s un - paired T - * p < 0 . 05 .

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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 of Fam134b - 2 from growth plate lysates of mice with indicated genotypes . β - actin was used as loading control . N = 6 ( wt mice ) N = 4 ( Fgfr3 / 4 dKO mice ) were analyzed . Bar graph showed of Fam134b - 2 normalized to β - actin . Mean + / - standard error of mean ( ) . Student ' s un - paired T - * p < 0 . 05 .

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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 of CLIMP - 63 of femur growth plate sections from mice with indicated genotypes . Scale bar 10μm . Insets showed increased CLIMP - 63 in Fgfr3 / 4 dKO mice . Scale bar 5 μm . Hypert = hypertrophic chondrocytes ; pre - hypert = pre - hypertrophic chondrocytes ; prolif = proliferating chondrocytes .

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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 of growth plate chondrocytes from mice with indicated genotypes . Arrows indicated 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 compartments biological processes regulated by FGF18 in a Fam134b - dependent manner . Score : Student ' s T - difference between wild type Fam134b ∆ LIR . N = 3 biological replicates / treatment / genotype were analysed . FDR < 0 . 05 .

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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 of chondrocytes with indicated genotypes treatments . Fam134b - dependent secreted are shown N = 3 biological replicates / treatment / genotype were analysed . FDR < 0 . 05 .

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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 of Fam134b Lamp1 from medaka fish treated with FGF18 ( 50 ng / mL ) for 24h . Values were normalized to Hprt gene expressed as fold change relative to untreated fish . N = 5 biological replicates . Mean + / - standard error of mean ( ) . Paired Student ' s T - * * p < 0 . 005 ; * p < 0 . 05 .

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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 of Lamp1 ( lysosome ) PDI ( ER ) in medaka fish chondrocytes with indicated genotypes treated with FGF18 ( 50 ng / mL for 24h ) . Insets showed details of colocalization of Lamp1 PDI . Wheat germ agglutinin ( WGA ) was used to stain cartilage matrix . Representative of N = 3 fish / genotype / treatment . Scale bars 10 μm . Boxed were zoomed single merged channels are shown . Scale bars 2 μm .

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D. Co-immunofluorescence staining of Lamp1 (lysosome) and PDI (ER) in medaka fish chondrocytes with indicated genotypes treated with FGF18 (50 ng/mL for 24h). Insets showed details of colocalization of Lamp1 and PDI. Wheat germ agglutinin (WGA) was used to stain cartilage matrix. Representative images of N=3 fish/genotype/treatment. Scale bars 10 μm. Boxed area were zoomed and single and merged channels are shown. Scale bars 2 μm.


E . of PDI into Lamp1 positive lysosomes . Mean + / - standard error of mean ( ) of N = 10 / experiment were analyzed . of variance one way ( ANOVA ) p < 0 . 001 ; Sidak ' s multiple comparison * * * p < 0 . 0005 ; NS not significant .

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E. Quantification of PDI fluorescence intensity into Lamp1 positive lysosomes. Mean +/- standard error of the mean (sem) of N=10 cells/experiment were analyzed. Analysis of variance one way (ANOVA) p<0.001; Sidak's multiple comparison test ***p<0.0005; NS not significant.


F . Western blot of type II procollagen from a pool of medaka fish with indicated genotypes , showing procollagen accumulation in Fam134bmo fish . β - actin was used as loading control . Bar graph shows of type II procollagen normalized to β - actin . N = 3 biological replicates . Mean + / - standard error of mean ( ) . Student ' s un - paired T - * p < 0 . 05 .

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F. Western blot analysis of type II procollagen from a pool of medaka fish embryos with indicated genotypes, showing procollagen accumulation in Fam134bmo fish. β-actin was used as loading control. Bar graph shows quantification of type II procollagen normalized to β-actin. N=3 biological replicates. Mean +/- standard error of the mean (sem). Student's un-paired T-test *p<0.05.


G . Representative of medaka fish chondrocytes . Arrows indicated ER . Insets show ER enlargement in Fam134bmo chondrocyte . N = 3 fish / genotype were analyzed . Bar graph shows number of ER enlargement per in medaka fish with indicated genotypes . N = 29 ( Fam134bwt ) N = 37 ( Fam134bmo ) were analyzed . Mean + / - standard error of mean ( ) . Student ' s un - paired T - * * * p < 0 . 0005 . Scale bar 500 nm . N = nucleus , ER = Endoplasmic Reticulum .

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G. Representative TEM images of medaka fish chondrocytes. Arrows indicated the ER. Insets show ER enlargement in Fam134bmo chondrocyte. N=3 fish/genotype were analyzed. Bar graph shows number of ER enlargement per cell in medaka fish with indicated genotypes. N=29 cells (Fam134bwt) and N=37 cells (Fam134bmo) were analyzed. Mean +/- standard error of the mean (sem). Student's un-paired T-test ***p<0.0005. Scale bar 500 nm. N=nucleus, ER= Endoplasmic Reticulum.


H . Lateral ( top ) ventral ( bottom ) projections of medaka fish with indicated genotypes . Scale bar 1mm . Bar graphs show of total head of medaka fish model of Fam134bmo expressed as % relative to scramble . Mean + / - standard error of mean ( ) of at least n = 8 fish / genotype . Student un - paired T - * p < 0 . 05 .

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H. Lateral (top) and ventral (bottom) projections of medaka fish embryos with indicated genotypes. Scale bar 1mm. Bar graphs show quantification of total length and head size of medaka fish model of Fam134bmo expressed as % relative to the scramble. Mean +/- standard error of the mean (sem) of at least n=8 fish/genotype. Student un-paired T-Test *p<0.05.


Alcian blue ( cartilage ) ( I ) of scramble Fam134bmo medaka fish . Graph shows of Ethmoid plate ( EP ) , Palatoquadrate ( PQ ) , Ceratohyal ( CH ) , Paired Prootics ( PO ) , Ceretobranchials 1 to 5 ( CB1 to CB5 ) cartilage ( I ) in Fam134bmo scramble fish . Values were expressed as % relative to scramble ( 100 % red dotted ) . Mean + / - standard error of mean ( ) of n = 9 fish / genotype . Student unpaired T - * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 0005 . NS not significant .

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Alcian blue (cartilage) (I) staining of scramble and Fam134bmo medaka fish. Graph shows quantification of Ethmoid plate (EP), Palatoquadrate (PQ), Ceratohyal (CH), Paired Prootics (PO), Ceretobranchials 1 to 5 (CB1 to CB5) cartilage length (I) in Fam134bmo and scramble fish. Values were expressed as % relative to the scramble (100% red dotted line). Mean +/- standard error of the mean (sem) of n=9 fish/genotype. Student unpaired T-Test *p<0.05; **p<0.005; ***p<0.0005. NS not significant.


Alizarin Red ( bone ) ( J ) of scramble Fam134bmo medaka fish . Graph shows of Ethmoid plate ( EP ) , Palatoquadrate ( PQ ) , Ceratohyal ( CH ) , Paired Prootics ( PO ) , Ceretobranchials 1 to 5 ( CB1 to CB5 ) bone mineralization ( J ) in Fam134bmo scramble fish . Values were expressed as % relative to scramble ( 100 % red dotted ) . Mean + / - standard error of mean ( ) of n = 9 fish / genotype . Student unpaired T - * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 0005 . NS not significant .

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Alizarin Red (bone) (J) staining of scramble and Fam134bmo medaka fish. Graph shows quantification of Ethmoid plate (EP), Palatoquadrate (PQ), Ceratohyal (CH), Paired Prootics (PO), Ceretobranchials 1 to 5 (CB1 to CB5) bone mineralization (J) in Fam134bmo and scramble fish. Values were expressed as % relative to the scramble (100% red dotted line). Mean +/- standard error of the mean (sem) of n=9 fish/genotype. Student unpaired T-Test *p<0.05; **p<0.005; ***p<0.0005. NS not significant.


B . ARPI ( CSS , ENZA , ODM - 201 , 14449 ) decreased , while R1881 increased , SDHA SDHB subunit in both LNCaP LAPC4 . PSA is used as a positive control .

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B. ARPI (CSS, ENZA, ODM-201, 14449) decreased, while R1881 increased, SDHA and SDHB subunit protein levels in both LNCaP and LAPC4 cells. PSA is used as a positive control.


D . SDH repression during acute phase of ENZA stress increased transcription of different AR - regulated genes without altering AR mRNAs in LNCaP . Data information : ENZA : enzalutamide . Data shown as mean ± SD of three independent experiments . Statistical was performed using two unpaired Student ' s t - * , p < 0 . 05 * * , p < 0 . 01 compared between groups .

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D. SDH repression during acute phase of ENZA stress increased transcription of different AR-regulated genes without altering AR mRNAs levels in LNCaP cells. Data information: ENZA: enzalutamide.Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using two tailed unpaired Student's t-test *, p<0.05 and **, p<0.01 compared between groups.


E - F . Overexpression of SDHA SDHB subunits decreased AR ( E ) AR transactivation ( F ) in LNCaP . Data information : ENZA : enzalutamide . Data shown as mean ± SD of three independent experiments . Statistical was performed using one - way ANOVA followed by Tukey ' s * , p < 0 . 05 * * , p < 0 . 01 compared between groups .

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E-F. Overexpression of SDHA and SDHB subunits decreased AR protein levels (E) and AR transactivation (F) in LNCaP cells. Data information: ENZA: enzalutamide.Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey's test *, p<0.05 and **, p<0.01 compared between groups.


C . SDH - repressed LNCaP were able to maintain higher proliferation rate particularly during ENZA stress with or without Hsp27 silencing in real time proliferation . Data information : ENZA : enzalutamide . Data shown as mean ± SD of three independent experiments . Statistical was performed using one - way ANOVA following Tukey ' s for panels C ( end points ) . * , p < 0 . 05 , * * , p < 0 . 01 * * * , p < 0 . 001 compared between

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C. SDH-repressed LNCaP cells were able to maintain higher proliferation rate particularly during ENZA stress with or without Hsp27 silencing in real time cell proliferation assay. Data information: ENZA: enzalutamide. Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA following Tukey's test for panels C (end points) . *, p<0.05, **, p<0.01 and ***, p<0.001 compared between groups


E . Treatment with p - p38 inhibitors : SB203580 ( 10 µM ) or Genistein ( 20 µM ) for 24 h reduced AR upregulation induced by SDH repression in LNCaP ( left panel ) . Bar graph in right panel shows band normalized to loading control . Data information : ENZA : enzalutamide . Data shown as mean ± SD of three independent experiments . Statistical was performed using one - way ANOVA following Tukey ' s for panels E . * , p < 0 . 05 , * * , p < 0 . 01 * * * , p < 0 . 001 compared between

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E. Treatment with p-p38 inhibitors: SB203580 (10 µM) or Genistein (20 µM) for 24 h reduced AR upregulation induced by SDH repression in LNCaP cells (left panel). Bar graph in the right panel shows band intensity normalized to loading control. Data information: ENZA: enzalutamide. Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA following Tukey's test for panels E . *, p<0.05, **, p<0.01 and ***, p<0.001 compared between groups


F . . Treatment with 5 µM STO - 609 for 24 h in LNCaP inhibited AR upregulation induced by SDHA / SDHB subunit silencing .

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F.. Treatment with 5 µM STO-609 for 24 h in LNCaP cells inhibited AR upregulation induced by SDHA/SDHB subunit silencing.


G . Thapsigargin , DMM , DMS ENZA treatment for indicated time points increased intracellular calcium flux in LNCaP as visualized with calcium sensitive Fluo4 - AM probe under confocal microscope .

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G. Thapsigargin, DMM, DMS and ENZA treatment for the indicated time points increased intracellular calcium flux in LNCaP cells as visualized with calcium sensitive Fluo4-AM probe under confocal microscope.