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. 2020 Mar 11;21(5):e48901. doi: 10.15252/embr.201948901

Figure 5. Malfunctioned ciliogenesis observed during TMEM135 depletion is not associated with IFT20.

Figure 5

  1. RPE1 cells were transfected with siRNAs as indicated, followed by 24‐h incubation in serum‐starved media. Cells were then and subjected to fractionation, and Western blot for IFT20, the Golgi marker GM130, membrane marker UBXD8, and nuclear marker CREB.
  2. Efficiency of IFT20 knockdown by Western blot.
  3. Cells were transfected with siRNAs as indicated, followed by incubation in serum‐starved media for 24 h, and immunostained for ARL13B (red). Scale bar, 10 μm. The bar graph represents the quantification of the percentage of ciliated cells. Data represent mean ± SD (n = 3 experiments), and 250 cells were scored per condition per experiment; *P < 0.05, Student's t‐test.
  4. Cells were transfected as shown in (C), and immunostained for Rab8 and γ‐tubulin, followed by quantification of the percentage of cells with Rab8 localized to the centriole. Data represent average (n = 2 experiments).
  5. Cells were transfected with siRNAs as indicated, followed by transfection with Flag‐IFT20, incubated in serum‐starvation media for 12 h, and immunostained for ARL13B. Representative fluorescent images of Flag‐IFT20 (green), ARL13B (red), and DAPI (blue) are shown. Scale bar, 10 μm.
  6. Quantification of the percentage of ciliated cells with both the Flag‐IFT20 and ARL13B localized in the cilium. Data represent mean ± SD (n = 3 experiments), and 150 Flag‐positive cells were scored per condition per experiment; *P < 0.05, Student's t‐test.
  7. Cells were transfected with siRNAs as indicated, followed by further transfection with CA‐Rab8, incubated in serum‐starvation media for 12 h, and immunostained for acetylated tubulin. Representative fluorescent images of GFP‐Rab8 Q67L (green), acetylated tubulin (red), and DNA (blue) are shown. Scale bar, 10 μm.
  8. Quantification of the percentage of GFP‐positive ciliated cells (only those cilia having both GFP‐Rab8 and acetylated tubulin on cilium were considered for quantification). Data represent mean ± SD (n = 3 experiments), and 200 GFP‐positive cells were scored per condition per experiment, *P < 0.05, Student's t‐test.

Source data are available online for this figure.