Figure 1

VDR knockdown in MDA-MB-231 cells reduces cell growth in vitro in a ligand-independent manner. (a,b) Compared to non-target controls (MDA-NT, cells transfected with non-target RNA), VDR mRNA (a) and protein (b) expression were knocked down by approximately 80% in MDA-VDR-KD cells at 24 h post plating. **P<0.01. (c) Treatment of MDA-NT and MDA-VDR-KD cells with 10⁻⁸ mol·L⁻¹ 1,25(OH)₂D₃ for 8 h increased CYP24 expression by 80-fold in MDA-NT cells with no appreciable response in MDA-VDR-KD cells. **P<0.01 compared to baseline. (d–i) Culture of MDA-NT and MDA-VDR-KD cells under ligand-free conditions: Compared to NT cells, cell growth (d) and cell proliferation (Ki67 immunoreactivity, (e) as well as Cyclin D1 mRNA of VDR-KD cells (f) were reduced by 40%, 36% and 50%, respectively. However, apoptosis was increased 6-fold (g) and Caspase 3 mRNA and protein was increased by 50% (h,i). Treatment of MDA-NT cells with 10⁻⁸ mol·L⁻¹ 1,25(OH)₂D₃ reduced both cell growth (d), Ki67 positivity (e) and Cyclin D1 mRNA expression (f) and induced a 2-fold increase in cell apoptosis (g) as well as Caspase 3 mRNA and protein expression (h,i) compared to untreated MDA-NT cells. In contrast, the same treatment had no effect on the growth or apoptosis rates of MDA-VDR-KD cells (d–i). Asterisks denote significant difference from untreated MDA-NT cells (*P<0.05; **P<0.01). In vitro experiments were performed in triplicate and repeated at least three times. Results shown are from a single representative experiment. Data are expressed as mean±s.e.m. (n=3).