Fig. 2

USP8 activity is essential for suppression of ciliogenesis and stabilization of trichoplein protein. a–c TetOn-RPE1 FLAG-USP8 cell lines (WT, S718A, and C786S) transfected with control or USP8 siRNA (#1) were cultured for 48 h in the presence or absence of doxycycline (Dox, 100 ng ml⁻¹). d–g Expression of FLAG-USP8 (WT, S718A, and C786S) was induced by treatment with Dox (100 ng ml⁻¹), and then subjected to serum starvation as shown in d. Representative confocal images of anti-acetylated-tubulin (green), anti-FLAG (red), and DAPI (blue) are shown in a and e. Insets are magnified images of dashed squares. Scale bars, 10 μm. Percentages of ciliated cells are shown as mean ± SD from three independent biological replicates (n > 200 each) in b and f. Immunoblotting analysis of trichoplein, USP8, FLAG and GAPDH are shown in c and g. Normalized intensities of trichoplein/GAPDH are shown as mean from three independent biological replicates. h FLAG-USP8 (WT or C786S) or GFP (as a control) were expressed by treatment with Dox (100 ng ml⁻¹) in TetOn-RPE1 cells, and the cells were treated with 200 nM cycloheximde-supplemented serum-starved medium for indicated times. Normalized intensities of trichoplein/GAPDH are shown as mean ± SD from three independent technical replicates. **p < 0.01, *0.01 < p < 0.05, n.s. not significant, two-tailed unpaired student’s t-tests