FIGURE 4.

Effect of cofactor silencing on the p21 mRNA response to 1α,25,(OH)₂D₃. MDA-MB453 cells were transfected for 24 h with siRNA oligonucleotides against the genes MED1, LSD1, HDAC4, NCoR1, HDAC3, CBP, or with a non-targeted siRNA (siCtrl), and subsequently either not stimulated (A) or stimulated (B) for the indicated times with 10 nm 1α,25(OH)₂D₃. Real-time quantitative PCR was used to determine the mRNA expression of indicated siRNA target genes (A). Remaining expressions were calculated in reference to samples transfected with a non-targeted siRNA (siCtrl). Real-time quantitative PCR was performed to measure the time-dependent mRNA expression of the p21 gene relative to the control gene RPLP0 (B). Fold inductions were calculated in reference to vehicle control that had been transfected with non-targeted siRNA (siCtrl). Data points indicate the means of at least three independent cell treatments, and the bars represent standard deviations. For B, a two-tailed Student's t test was performed to determine the significance of the effects of the specific siRNAs in reference to control siRNA (^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001).