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. 2019 Oct 16;218(12):4030–4041. doi: 10.1083/jcb.201811136

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© 2019 Wang et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

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Figure 1. — Depletion of Rab7 promotes ciliogenesis. (A) Rab7 knockdown using three individual siRNAs induced spontaneous ciliogenesis in cycling cells (+serum). RPE-1 cells transfected with the indicated siRNAs were stained with acetylated α-tubulin (Ac-tub, green) and ADP ribosylation factor–like GTPase 13B (Arl13b, red). Nuclei were stained with Hoechst (blue). Scale bars, 10 µm (main image) and 2 µm (magnified regions). (B) The percentage of cells with primary cilia in A were quantified. Data are means ± SD of four independent experiments. Student’s t test was performed. **P < 0.01, ***P < 0.001. Cell lysates were immunoblotted with the indicated antibodies. GAPDH was used as a loading control. (C) Rab7 knockdown promoted cilia elongation in quiescent (−serum) RPE-1 cells. The length of primary cilia was measured. CP110 was used as a negative control. Data are means ± SD. Mann-Whitney rank sum test was performed. ***P < 0.001. Cell lysates were immunoblotted with the indicated antibodies. GAPDH was used as a loading control. (D) Representative images of C. RPE-1 cells transfected with the indicated siRNAs were stained with acetylated α-tubulin (green) and γ-tubulin (red). Acetylated α-tubulin labels cilia and γ-tubulin labels centrosomes. Nuclei were stained with Hoechst (blue). Scale bars, 10 µm (main image) and 2 µm (magnified regions). (E and F) Spontaneous ciliogenesis induced by Rab7 knockdown in RPE-1 cells were rescued by Flag-Rab7-WT and the Q67L mutant, but not by the T22N mutant. RPE-1 cells transfected with the indicated siRNAs and plasmids were cultured in serum medium for 48 h. WT, wild-type Rab7; T22N, dominant negative mutant of Rab7; Q67L, dominant active mutant of Rab7. All constructs were RNAi resistant. Cells were stained with Flag (green) and Arl13b (red). Nuclei were stained with Hoechst (blue). Scale bar, 5 µm. Data are means ± SD of three independent experiments. ***P < 0.001. (G and H) Prolonged cilia length induced by Rab7 knockdown in RPE-1 cells was rescued by Flag-Rab7-WT and the Q67L mutant, but not by the T22N mutant. RPE-1 cells transfected with the indicated siRNAs and plasmids were cultured in serum-free medium for 48 h. All constructs were RNAi resistant. Cells were stained with Flag (green) and Arl13b (red). Nuclei were stained with Hoechst (blue). Scale bar, 5 µm. Data are means ± SD. Student’s t test was performed. ***P < 0.001.