Fig 1: Identification of ARHGAP11A as a cell cycle-dependent mobility- controlling molecule.(A) Fucci signal-based microarray analyses. Fucci-positive HCT116 cells were separated into Fucci-green (mAG)- and Fucci-red (mKO2)- positive cells by FACS. mRNA was extracted from these cells and compared by microarray analysis (two dye-swap experiments, giving four independent microarray analyses). (B) In total, 2,023 probes (1,656 genes) showed >twofold changes in expression (Table S1) (P>0.05). Of them, Arhgap11a was highly ranked, and all three probes for Arhgap11a were among the top-ranked probes for RhoGAPs. The three probes were specific for the indicated potions of the Arhgap11a mRNA (left). The three marked dots (#1, #2, #3) in the scatter plots represent fold changes (right). (C) Cell cycle-dependent expression of Arhgap11a mRNA was confirmed by qPCR. The expression data were normalized to Gapdh (n = 3). (D) Cell cycle-dependent expression of Arhgap11a proteins. (Right) Flow cytometric analyses of Fucci-expressing HCT116 cells. Cell cycle profiles were color-coded: G1, red; early S, yellow; and S/G2/M, green (upper right). DNA contents were measured by Hoechst33342 fluorescence of (lower right), confirming that Fucci signals accurately represent cell cycle levels (n = 3). (Left) Cell cycle-dependent expression of ARHGAP11A and cell cycle markers, as determined by Western blotting (n = 3). (E) Time-dependent expression of Arhgap11a during progression of the cell cycle from G1 to S/G2/M. Fucci-red (mKO2)-positive HCT116 cells were sorted using a FACSAria cell sorter and were cultured for the indicated periods of time. Flow cytometry analyses (upper) and ratios of Fucci colors (left) are shown for each time point. (Right) Relative expression of Arhgap11a was examined by qPCR (n = 6). Data were analyzed by one-way ANOVA (p = 0.0001) and Bonferroni's multiple comparison test (*** p<0.01). (F) Cell cycle-dependent Arhgap11a expression in various human colon cancer cell lines. Fucci was introduced into different human colon cancer cell lines (HCT116, DLD1, HT29, and KM12SM). In all of the cell lines, Arhgap11a expression was significant higher in S/G2/M (green) than in G1 cells (red). (G) A chromatin immunoprecipitation (ChIP) assay with an anti-E2F1 antibody showed that E2F1 bound to the putative E2F-binding site in the Arhgap11a promoter (n = 3). (H) Luciferase reporter assay of the Arhgap11a promoter region (-500 bp), including the E2F-binding site (GTTTCGCGC) at -20 bp from the transcription starting point. Co-transfection with E2F1 enhanced transcriptional activity, whereas simultaneous expression of Rb blocked it. Values for luciferase activity were normalized across each experiment and, to control for differences in transfection efficiency, to ß-galactosidase.