Figure 2A Immunoblot analysis of RAB35 depletion in hTERT-RPE1 cells transfected with two independent siRNAs targeting RAB35 (RAB35-1, RAB35-2) or a non-targeting siRNA control (Neg). 24 h after transfection, cells were serum-starved for further 48 h. β-tubulin served as a loading control. B Quantification of ciliation of hTERT-RPE1 cells treated and using acetylated tubulin staining as a cilia marker. Data are mean ± S.E.M. of 4 independent experiments (n ≥ 50 cilia per experimental condition). C, D hTERT-RPE1 cells and hTERT-RPE1 cells stably expressing siRNA-resistant GFP-RAB35 were treated and stained for GFP, acetylated tubulin (acetyl. tub.) and DNA. Representative images in (C) of cells treated with Neg or RAB35 siRNA. Regions within white boxes shown at higher magnifications to the right. Scale bars, 10 µm. Cilia length quantifications in (D) are shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment of three is shown (n ≥ 50 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (* P < 0.05, ** P < 0.01, *** P < 0.001, n.s.: non-significant; P-values: hTERT-RPE1-Neg vs. hTERT-RPE1-RAB35-1 P < 0.0001, hTERT-RPE1-Neg vs. hTERT-RPE1-RAB35-1 P < 0.0001, hTERT-RPE1-RAB35-1 vs. GFP-RAB35-RAB35-1 P = 0.0249, hTERT-RPE1-RAB35-2 vs. GFP-RAB35-RAB35-2 P = 0.0056) E Immunoblot analysis of Rab35 depletion in IMCD3 cells transfected with pool of siRNAs targeting mouse Rab35 or a non-targeting siRNA control (Neg). 24 h after transfection, cells were serum-starved for further 48 h. GAPDH served as a loading control. F Quantification of ciliation of IMCD3 cells treated and using acetylated tubulin staining as a cilia marker. Data are mean ± S.E.M. of 3 independent experiments (n ≥ 50 cilia per experimental condition). G-H Immunofluorescence of IMCD3 representative images treated cells are stained for acetylated tubulin (acetyl. tub.) and DNA. Cilia length quantification in (H) is shown as box-and-whisker plots and is the result of 3 independent experiments (n ≥ 50 cilia per experimental condition). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. Statistical significance according to unpaired t-test with Mann-Whitney test (*** P = 0.0002) I Immunoblot analysis of NIH3T3 wild type (WT) and Rab35 knockout (KO) cell lines. β-tubulin served as a loading control. NIH3T3 WT and Rab35 KO cell lines were serum-starved for 24h and stained for acetylated tubulin (acetyl. tub.) and DNA. Quantification of ciliation in (J). Data are mean ± S.E.M. of 3 independent experiments (n ≥ 100 cilia per experimental condition). NIH3T3 WT and Rab35 KO cell lines were serum-starved for 24h and stained for acetylated tubulin (acetyl. tub.) and DNA. Representative images in (K). Regions within white boxes shown at higher magnifications at the bottom. Scale bars, 10 µm. Cilia length quantifications in (L) are shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment of three is shown (n ≥ 100 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (**** P < 0.0001).