Figure 1

Cilia, BBS4 and BBS2 knockdown result in a reduction of intracellular and secreted FSTL1. (A) hTERT-RPE1 cells were transfected with control siRNA (Cneg) or siRNA BBS4 to produce BBS4 knock-down (BBS4). Secreted FSTL1 and intracellular FSTL1 levels were analyzed in cell culture media (SN) and whole cell lysates (WCL) respectively by Western blot using anti-FSTL1. Full-length blots are shown in Figure S7A. (B) hTERT-RPE1 cells were transfected with siRNA to target FSTL1 and IFT88 or siRNA Cneg. Secreted FSTL1 and intracellular FSTL1 were analyzed in SN and WCL respectively by Western blot using anti-FSTL1. In both A and B Ponceau S staining of an unspecific blotted protein and anti-α tubulin were used as loading controls to normalize FSTL1 abundance. Full-length gels are shown in Figure S7D. (C) Quantitative representation of densitometry readings of 3 combined experiments shown in (A) and (B). BBS4-KD cells show reduced levels of both intra- and extracellular FSTL1. (D) hTERT-RPE1 cells were transfected with siRNA BBS2 or siRNA Cneg and secreted FSTL1 was analyzed by Western blot anti-FSTL1 and Ponceau S staining was used to normalize. The Cneg and BBS2 KD set of lanes were cropped from the same blot. Full-length blots are shown in Figure S7E. (E) Densitometry readings of Western blot shown in (D). The results shown are representative of two independent experiments. (F) SN of hTERT-RPE1 cells transfected with siRNA BBS4 were analyzed by Western blot with anti-Laminin and Ponceau S staining to normalize (upper panel). Densitometry readings of the western blot showed that BBS4-KD does not affect Laminin secretion (lower panel). The full-length blot is shown in Figure S7F. (G) qRT-PCR was performed to analyze FSTL1 gene expression in hTERT-RPE1 cells transfected with the following siRNAs: Cneg, BBS4, FSTL1, IFT88 and BBS2. FSTL1 mRNA levels are represented as % expression relative to control siRNA (Cneg)-transfected cells. FSTL1 expression is reduced by BBS2 and BBS4 KD cells as well as by IFT88/cilia knockdown. Data from at least two independent experiments, designed with biological duplicates, and three technical replicates were normalized relative to Cneg siRNA transfected cells and combined for the analysis. In all cases, error bars represent standard deviation. **: P = 0,001–0,01; ***: P = 0,0001–0,001 and ****: P < 0,0001, ANOVA or t-test.