MiT/TFE factors control ER-phagy via transcriptional regulation of FAM134B (2020)
Figure 3

Figure 3. FGF signaling induces ER-phagy via FAM134B.

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.

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

D. Heat-map of lysosomal and autophagy genes expression in RCS chondrocytes with indicated genotypes (ctrl=wild type), treated with vehicle (5% ABS) and FGF18 (50 ng/mL) for 16 hours. FDR <0.05. N=3 biological replicates/treatment were analyzed. In green: down-regulated, in red: up-regulated gene expression.

E. qRT-PCR analysis of ER-phagy receptors in RCS chondrocytes. Gene expression was analyzed after FGF18 (50 ng/mL) treatment for 16 hours. Fold change values were relative to vehicle (5% ABS) and normalized to Cyclophilin gene. Mean +/- standard error of the mean (sem) of N=3 biological replicates/treatment. Student's paired T-test **p<0.005; *p<0.05.

F. (top) Representative model of Fam134b∆LIR protein. LIR= LC3-Interacting-Region. (bottom) Western blot analysis of Fam134b in chondrocytes with indicated genotypes (ctrl=wild type) treated with FGF18 (50 ng/mL) for 16 hours. Representative images of N=3 biological replicates/treatment. β-actin was used as loading control. Bar graph showed quantification of Fam134b band intensity normalized to β-actin. Mean +/- standard error of the mean (sem). Student's paired T-test, *p<0.05.

G. Immunofluorescence staining of CLIMP63 (green) and lysosomes (TMEM192-HA, red) in RCS chondrocytes with indicated genotypes (ctrl=wild type) upon FGF18 treatment (50 ng/mL for 16h). BafA1 (100 nM; 4 hours) was used to inhibit lysosomal degradation; scale bar 20 μm. Insets show magnification of CLIMP63 accumulation into lysosomes; scale bar 2 μm. Quantification of CLIMP63 fluorescence intensity into TMEM192-HA decorated lysosomes. Mean +/- standard error of the mean (sem) of N=3 biological replicates. n=10 cells/experiment were analyzed. Analysis of variance one way (ANOVA) p=1.55e-7; Tukey's post hoc test ***p<0.0005; NS not significant.

H. EATR assay in RCS with indicated genotypes (ctrl=wild type) showing % of cells with acidified ER by FACS analysis. FGF18 was used at 50 ng/mL for 16 hours. Mean +/- standard error of the mean (sem) of N=4 biological replicates/treatment/genotype. Analysis of variance one way (ANOVA) p<0.0001; Tukey's post hoc test ***p<0.0005; NS not significant.