PCIF1 suppresses ciliogenesis in RPE-1 cells. (A–D) RPE-1 cells were treated with control or PCIF1 siRNAs for 48 h under normal culture conditions (DMEM/F12 supplemented with 10% FBS) and then applied for Western blotting or immunofluorescence analysis. (E–G) RPE-1 cells treated with the indicated siRNAs for 24 h were transfected with control or PCIF1 plasmid for another 36 h under normal culture conditions and then subjected to Western analysis or immunofluorescence. (H–K) RPE-1 cells transfected with the indicated plasmids for 48 h were treated with serum starvation for another 24 h and then applied for western analysis or immunofluorescence. Western blotting analysis of PCIF1 protein (A, E, and H). Actin is the internal control. Immunofluorescence images of RPE-1 cells with anti-ARL13B (green) and γ-tubulin (red) antibodies (B, F, and I). DNA was stained with DAPI (blue). Cilia were indicated by white arrows. Scale bar, 10 μm. Quantification of the percentage of ciliated cells (C, G, and J). Cilia length is measured by ImageJ (D and K). Quantitative analyses are depicted in SuperPlots, with each color representing an independent biological replicate. In the SuperPlots, smaller symbols indicate the ciliation percentage for each field of view or the cilia length of individual cells, while larger symbols represent the mean value calculated for each replicate. All error bars represent means ± SD. Student’s t test, **P < 0.01, ***P < 0.001, ns, not significant. Source data are available for this figure: SourceData F1.