Fig 2: OFD1 ablation-elicited cell cycle arrest is reversed by oncogene activation and RB inactivation in transformed cells.a Representative images of Ki67 (magenta) or ARL13B (magenta) staining in Tet-inducible EGFP-OFD1-expressing RPE1 cells with indicated titration of Doxycycline and indicated siRNAs for 72 h. b Quantitation of cells positive for ARL13B or Ki67 staining in (a). Data shown represent mean values ± SD percentage of cells from triplicate samples. 300 cells examined over three independent experiments, P = 0.0019; P = 0.1191; P = 0.0024; P = 0.0214; P = 3.21 × 10-5; P = 0.00408; P = 0.00412; P = 0.0402, two-tailed unpaired student’s t-test. Cilia are marked by arrowheads. c Normal or cancer cells were transfected with OFD1 siRNA and immunostained for Ki67 to mark proliferating cells. Cell lysates of RPE1 cells treated as indicated were immunoblotted for OFD1 and ß-tubulin. Data shown represent mean ± SD percentage of cells positive for Ki67 are from triplicate samples. 300 cells examined over three independent experiments. d Normal cells and cancer cells were treated with 120 µM CK-666 or 120 nM Cyto D and immunostained for Ki67. Data shown represent percentage of cells positive for Ki67 are from triplicate samples. 300 cells examined over three independent experiments. e–g RNAi knockdown of all three members of the RB protein family, RB1, RBL1, and RBL2, in human RPE1 cells abolishes OFD1 loss-induced cell cycle arrest but not cilia formation. e Immunoblot analysis of RPE1 cell lysates for the indicated proteins is shown. f Representative immunofluorescence staining of Ki67 (red) and ARL13B (green) in RPE1 cells with indicated treatments. Cilia are marked by arrowheads. g Quantitation of ARL13B or Ki67-positive cells in f. Data shown represent mean value ± SD, 300 cells examined over three independent experiments, P = 0.002, two-tailed unpaired student’s t-test. h Representative images of Ki67 (red) and ARL13B (green) staining of RPE1 cells transfected with control siRNA or RB siRNA for 72 h. Cells were treated for 48 h with or without CK-666 24 h after siRNA transfection. Cilia are marked by arrowheads. i Quantitation of cells positive for ARL13B or Ki67 staining in h. Data shown represent mean ± SD percentage of cells from triplicate samples. 300 cells examined over three independent experiments, P = 0.004, two-tailed unpaired student’s t-test. All data shown represented as mean values ± SD, error bar was defined as SD.