Figure 3

Depletion of RUNX2 has a modest mitogenic effect in MDA-MB-231 cells. (A) RUNX2 transcript levels in MDA-MB-231 cells, which were either treated with siRUNX2 or control non-silencing RNA (si-control), were measured using qRT-PCR analysis and normalized with GAPDH. (B) RUNX2 knockdown MDA-MB-231 cells results in increased expression of cyclin D1 and marginally decreased expression of p21 relative to GAPDH as measured using qRT-PCR analysis. (C, D) Protein levels detected by western blotting (C) and mRNA expression by qRT-PCR (D) was examined in MDA-MB-231 derived cell lines that were transduced with either shRUNX2 or empty vector control retroviruses. RNA was harvested from cells selected with puromycin (1 μg/ml) for two weeks. ShRNA mediated knock-down of RUNX2 mRNA (inset) dramatically reduced p21 mRNA levels (D), but putative compensatory changes may have prevented decreased accumulation of p21 protein (C). (E) Cell proliferation in MDA-MB-231 cells that either express shRUNX2 or contain empty vector (control) was measured by cell counting. Loss of RUNX2 has a modest positive effect on cell growth.