Figure 6. RUNX1 silencing deregulates breast cancer cell mitosis.

RUNX1 was knocked down in MCF7 cells either constitutively (a (left) and b) or conditionally upon treatment with dox (a (right), c,d g and h). Cells were maintained in medium supplemented with either complete serum (a,c,d,f–h) or CSS (b). (a,b) Cell cycle profiles were obtained by FACS analysis of propidium iodide-stained cells. In b, cells were treated with either vehicle control (EtOH) or estradiol (E2) for 48 h as indicated. *P<0.05 by t-test. (c) RT–qPCR analysis of the indicated Wnt/cell cycle-regulatory genes. Data are corrected for 18S RNA. (d) Luciferase assay of TOPFLASH or control FOPFLASH as a measure of β-catenin/TCF activity. (e) Expression levels of the indicated genes in RUNX1-mt (N=17) versus RUNX1-wt (N=389) ER⁺ tumours in the breast cancer cohort of TCGA. Significance of the differences was calculated using Mann–Whitney test. (f,g) Naive MCF7 (f) and MCF7/shRx1_RUNT^dox cells (g) were treated with 250 ng ml⁻¹ dox for 72 h and 2 nM docetaxel was added for the last 48 h as indicated. Percentages of cells in G1, S and G2/M are given as mean and s.e.m. values from three independent experiments. Representative plots are presented, with inset in g showing western blot analysis of cyclin B1. (h) MCF7/Rx1sh_RUNT^dox cells were synchronized as described in the ‘Methods' section at either G1/S or G2/M, or at the indicated time points. The cells were treated with 250 ng ml⁻¹ dox along with the release from the first thymidine block and extracts were subjected to western blot analysis of the indicated proteins. Quantitative data are mean±s.e.m. from three independent experiments. *P<0.05 by t-test.