Figure 2: Increased proliferation and expression of stem cell markers in RUNX1-depleted mammary epithelial cells in vitro and in vivo.

(a) MCF7 cells expressing nonspecific (NS) or the indicated RUNX1-targeting shRNAs were plated and their growth rate was assessed by MTT assays on days 3 and 6. (b) RUNX1 was silenced with shRx1_3′-UTR as in Fig. 1d,e, and was re-expressed from a dox-inducible vector as demonstrated by the western blot in the inset. Cell growth was assessed as in a and bars represent the increase in MTT values between day 3 and day 6. (c) MCF7 cells expressing a nonspecific shRNA (shNS) or shRx1_3′-UTR were subjected to RT–qPCR analysis of the indicated stem cell markers. Expression of Sox2 was also assessed by western blot analysis. (d) RT–qPCR analysis of SOX2 in MCF7 cells in which RUNX1 was silenced and then restored as in b. (e–g) Comparisons of SOX2 mRNA expression between (e) RUNX1-depleted versus control T47D cells; (f) mammary luminal epithelial cells from MMTV-Cre;Runx^f/f;R26Y versus control MMTV-Cre;Runx1^+/+;R26Y mice based on our microarray data in GSE47377 (ref. 13); (g) RUNX1-mutant (n=7) versus RUNX1-WT (n=202) breast cancer tumours based on our microarray database¹⁸, where boxes represent the 25th–75th percentile range, horizontal lines within boxes represent the median values and whiskers extend to the minimum and maximum values. Where applicable, data represent mean±s.e.m. of triplicate experiments. *P<0.05 by t-test (a–f) or by Mann–Whitney test (g).