Fig. 4

CMTM4 induces G2/M cell cycle arrest. a Annexin V/PI-staining indicated that overexpression of CMTM4 did not induce apoptosis of 786-O cells 72 h after infection. Shown is a representative result of three independent experiments. b The cell cycle was analysed 48 h after infection of 786-O cells by flow cytometry. Representative histograms (left) and the percentage of cells at the different phases (right) are shown. The data are expressed as the means ± SEM of three independent experiments. **, P < 0.01. c Western blotting analysis of cell cycle hallmarks in infected 786-O cells. β-actin was used as an internal standard. A representative result and the means of the relative intensities of the target proteins averaged for the three independent experiments are shown with the SEM. ***, P < 0.001; ns, not significant. d RT-PCR of p21 at 24 h after infection of 786-O cells. GAPDH was used as an internal standard. The grey density of the target bands was analysed by ImageJ software (National Institutes of Health, Bethesda, Maryland, U.S.) and normalised to the grey density of GAPDH. The average relative grey density with the SEM is shown from three independent experiments. Knockdown of CMTM4 reduced the G2/M phase accumulation (e) and p21 expression at both the protein (f) and mRNA levels (g). **, P < 0.01; and ***, P < 0.001