Rab35 controls cilium length, function and membrane composition (2019)
Figure 8

Figure 8. Rab35 depletion disrupts the ciliary localisation of Smoothened

A-C hTERT-RPE1 cells transfected with indicated siRNAs were serum-starved for 48 h and treated with SMO agonist (SAG) or vehicle control (DMSO) for the last 24 h. Cells were stained for SMO, acetylated tubulin and DNA. Representative images are shown in (A). Regions within white boxes shown at higher magnifications to the right. Scale bars, 10 µm. Graph in (B) shows the percentages of SMO-positive (SMO+) cilia. Data are mean ± S.E.M. of 5 independent experiments. Statistical significance according to ANOVA followed by Bonferroni post-hoc test (*** P < 0.001; P-values: Neg+SAG vs. RAB35-1+SAG P < 0.0001, Neg+SAG vs. RAB35-2+SAG P = 0.0033) (C) Box-and-whisker plots show quantification of the average SMO intensity in the ciliary area marked by acetylated tubulin staining. All cilia: cilia identified with acetylated tubulin staining. SMO+ cilia: cilia with discernible SMO localisation. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n ≥ 30 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (* P < 0.05, *** P < 0.001; P-values: Neg+SAG vs. RAB35-1+SAG (all cilia) P < 0.0001, Neg+SAG vs. RAB35-2+SAG (all cilia) P = 0.0024, Neg+SAG vs. RAB35-1+SAG (SMO+ cilia) P < 0.0007, Neg+SAG vs. RAB35-2+SAG (SMO+ cilia) P = 0.0205)

D-F For co-depletion experiments, hTERT-RPE1 cells were first transfected with Neg or RAB35 siRNA. After 24h, a second transfection with Neg or ARL13B siRNA was performed and cells were treated as in (A). Representative images are shown in (D). Regions within white boxes shown at higher magnifications as insets. Scale bars, 10 µm. Graph in (E) shows the percentages of SMO-positive cilia. Data are mean ± S.E.M. of 5 independent experiments. Statistical significance according to ANOVA followed by Bonferroni post-hoc test (* P = 0.0151, n.s.; non-significant). Box-and-whisker plots in (F) show quantification of ciliary SMO intensity. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n ≥ 30 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (* P = 0.0142, n.s.; non-significant).

G, H Ciliary SMO intensity quantification in hTERT-RPE1 cells transiently expressing GFP, wild type ARL13B-GFP (WT) or the ciliary-targeting defective mutant ARL13B-V358A. Cells were serum-starved and treated with SAG for 24 h and stained for SMO, acetylated tubulin (acetyl. tub.) and DNA. Representative images are shown in (G). Higher magnification images of the cilia region are shown at the bottom. Scale bars; 10 µm. Box-and-whisker plots in (H) show quantification of ciliary SMO intensity. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n ≥ 25 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (** P = 0.0046).