Figure 2

Lysosome inhibition rescues the FSTL1 degradation induced by BBS4 knockdown. (A) hTERT-RPE1 cells were transfected with siRNA Cneg, siRNA IFT88 or siRNA BBS4 for 48 hours and incubated for 6 hours with MG132. Cell lysates were analyzed by Western blot anti-FSTL1, anti α tubulin and anti-Ubiquitin. Full-length blots are shown in Figure S8A. (B) hTERT-RPE1 cells were transfected with siRNA Cneg, siRNA BBS4 (top panel) or siRNA IFT88 (bottom panel; for each condition, lanes cropped from the same gel are shown) during 24 hours and incubated with chloroquine for an additional 24 hours. Full-length blots are shown in Figure S8B. (C-F) Quantification of experiment shown in (B). Densitometry readings were used to calculate ratios of FSTL1/Tubulin to quantitate intracellular FSTL1 and FSTL1/Ponceau S to measure secreted FSTL1. Chloroquine treatment rescued intracellular levels of FSTL1 without affecting its extracellular abundance. Results shown are representative of three independent experiments performed with biological triplicates or duplicates as shown. Error bars represent standard deviation and ns: P > 0.05; *: P = 0,01–0,05; **: P = 0,001–0,01; ***: P = 0,0001–0,001, ANOVA test.