MiT/TFE factors control ER-phagy via transcriptional regulation of FAM134B (2020)
Figure 8

Figure 8. FAM134B is required for protein secretion in RCS and skeletal growth in Medaka.

A. Scatter plot of cellular compartments and biological processes regulated by FGF18 in a Fam134b-dependent manner. Full list can be found in table 7 and 8. 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, full list can be found in table 9. N=3 biological replicates/treatment/genotype were analysed. FDR <0.05.

C. qRT-PCR analysis of Fam134b and Lamp1 from medaka fish embryos treated with FGF18 (50 ng/mL) for 24h. Values were normalized to Hprt gene and expressed as fold change relative to untreated fish. N= 5 biological replicates. Mean +/- standard error of the mean (sem). Paired Student's T-test **p<0.005; *p<0.05.

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

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

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

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

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

I,J. Alcian blue (cartilage) (I) 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) cartilage length (I) and 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.

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