Figure 3A Representative images of hTERT-RPE1 cells transiently expressing wild type (WT), GDP-bound (S22N) or GTP-bound (Q67L) GFP-tagged RAB35. Cells were serum-starved for 24 h and stained for polyglutamylated tubulin (polyglu. tub.) and DNA. Higher magnification images of the cilia region shown in smaller panels. Scale bars; 10 µm. Quantification of GFP-RAB35 ciliary localisation (B) in hTERT-RPE1 cells transiently expressing indicated GFP-RAB35 constructs or GFP. Data are mean ± S.E.M. of 3 independent experiments. Statistical significance according to ANOVA followed by Bonferroni post-hoc test ( ** P < 0.01; P-values: (B) P = 0.0015 Quantification of ciliation (C) in hTERT-RPE1 cells transiently expressing indicated GFP-RAB35 constructs or GFP. Data are mean ± S.E.M. of 3 independent experiments. Statistical significance according to ANOVA followed by Bonferroni post-hoc test ( ** P < 0.01; P-values: (C) P = 0.0062). Quantification of ciliary length in hTERT-RPE1 cells transiently expressing indicated GFP-RAB35 constructs or GFP. Cilia length quantification in (D) is shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out 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-values: GFP vs. GFP-RAB35-S22N P = 0.0015, GFP vs. GFP-RAB35-Q67L P = 0.0158) Analysis of cilia length in hTERT-RPE1 cells depleted of GEF DENND1B) and GAP (TBC1D10A regulators of RAB35. Cells transfected with indicated siRNAs were serum-starved for 48 h and stained for acetylated tubulin (acetyl. tub.) and DNA. Representative images are shown in (E). Regions within white boxes shown at higher magnifications to the right. Scale bars; 10 µm. Analysis of cilia length in hTERT-RPE1 cells depleted of GEF (DENND1A, DENND1B) and GAP (TBC1D10A, TBC1D10B) regulators of RAB35. Cells transfected with indicated siRNAs were serum-starved for 48 h and stained for acetylated tubulin (acetyl. tub.) and DNA. Cilia length quantification in (F) is shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n ≥ 50 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (** P < 0.01, *** P < 0.001; P-values: Neg vs. DENND1B P = 0.0033, Neg vs. TBC1D10A P = 0.0008) G Quantification of ciliary localisation of GFP-RAB35 in hTERT-RPE1 cells stably expressing GFP-RAB35 treated Data are mean ± S.E.M. of 5 independent experiments. Statistical significance according to ANOVA followed by Bonferroni post-hoc test (* P < 0.05, *** P < 0.001; P-values: Neg vs. DENND1B P < 0.0001, Neg vs. TBC1D10A P = 0.0322). H-J Localisation of transiently expressed GFP-DENND1B (H) or GFP-TBC1D10A (I) in hTERT-RPE1 cells after 24 h serum starvation and staining with indicated antibodies. Higher magnification images of the cilia region are shown in smaller panels. Scale bars; 5 µm. Quantification of localisation to ciliary base or along axoneme in (J). Data are mean ± S.E.M. of 3 independent experiments (n ≥ 30 cilia per experimental condition).