MiT/TFE factors control ER-phagy via transcriptional regulation of FAM134B (2020)
Figure 5

Figure 5. FGF signaling regulates FAM134B transcriptional levels through TFEB and TFE3.

A. qRT-PCR analysis of Fam134b gene expression in chondrocytes with indicated genotypes (ctrl=wild type) treated with vehicle (5% ABS) or with FGF18 (50 ng/mL; 16 hours). Fold change values were relative to vehicle and normalized to Cyclophilin gene. Mean +/- standard error of the mean (sem) of N=3 biological replicates. Analysis of variance one way (ANOVA) p<0.0001: Tukey's post hoc test ***p<0.0005; NS not significant.

B. Western blot analysis of Fam134b protein in chondrocytes with indicated genotypes treated with vehicle (5% ABS) and FGF18 (50 ng/mL) overnight. β actin was used as loading control. Representative image of N=3 biological replicates. Bar graph showed quantification of Fam134b normalized to β-actin. Analysis of variance one way (ANOVA) p<0.0001; Sidak's multiple comparison test ***p<0.0005; **p<0.005 NS not significant.

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

D. Quantification of CLIMP63 fluorescence intensity into TMEM192-HA decorated lysosomes. Mean +/- standard error of the mean (sem) of N=3 biological replicates/treatment/genotype (ctrl=wild type). n=10 cells/experiment were analyzed. Analysis of variance one way (ANOVA) p=1.55e-7; Tukey's post hoc test ***p<0.0005; NS not significant.

E. EATR assay in chondrocytes with indicated genotypes (ctrl=wild type) showing % of cells with acidified ER measured by FACS. FGF18 was used at 50 ng/mL overnight. Mean +/- standard error of the mean (sem) of N=4 biological replicates. Analysis of variance one way (ANOVA) p<0.0001: Tukey's post hoc test ***p<0.0005; *p<0.05; NS not significant.

F,G Data plots show quantification of mCherry+ vesicles/cell (autolysosomes) (F) and mCherry+/GFP+ vesicles/cell (autophagosomes) (G) in wild type (ctrl) and TFEB;3KO cells treated with vehicle (veh) or FGF18. N=3 independent experiments. Mean +/- standard error of the mean (sem) of N=24 (wild type treated with 5%ABS, veh), N=30 (wild type treated with FGF18), N=27 (TFEB;3KO veh), N= 33 (TFEB;3KO FGF18) cells. Student un-paired T-Test ***p<0.0005; **p<0.005.

H. ChIP analysis of TFEB binding to Fam134b DNA in RCS cells transfected with TFEB-3XFLAG. Numbers in the CLEAR site (yellow box) refer to the distance [in base pairs] from the transcriptional start site (+1) of Fam134b-2 gene. Immunoprecipitated DNA was normalized to the input and plotted as relative enrichment over a mock control. Bar graph shows fold change enrichment; mean +/- standard error of the mean (sem) of N=3 independent experiments. Student un-paired T-Test **p<0.005.

I-J. Luciferase assays in RCS chondrocytes using as promoter a 0,7kb genomic Fam134b DNA fragment containing a wild type (FAM134B-WT) or a deleted (FAM134B-mut) version of the CLEAR site. TFEB plasmid transfection amount and FGF18 (50 ng/mL for 16h) treatments are indicated. Mean +/- standard error of the mean (sem) of N=3 biological replicates. Student paired T-test *p<0.05; **p<0.005.

K. Luciferase activity in wild type (ctrl) and TFEB;3KO RCS chondrocytes with indicated genotypes expressing the indicated Fam134b luciferase report plasmids and treated with FGF18 overnight (50 ng/mL) where indicated. Mean +/- standard error of the mean (sem) of N=5 biological replicates. Analysis of variance one way (ANOVA) p<0.0001: Sidak's multiple comparison test ***p<0.0005; NS not significant.