Down-regulation of IRF5 protein expression by siRNAs alters sensitivity to DNA damage. A. MCF-12A cells were incubated with transfection reagent alone (mock-transfected), control Lamin A/C siRNAs or 5 nM IRF5 siRNAs once (IRF5 siRNA1) or twice (IRF5 siRNA2), as described in the Materials and methods. Western blot analysis shows > 70% reduction of endogenous IRF5 proteins after normalization to β-actin levels. B. Cells were exposed to 5 Gy IR or the same dose plus IFN-γ (IR/γ) for 24 h. Percent of Annexin V-FITC stained positive cells is shown in the upper and lower right-hand quadrants. Representative histogram plots from three independent experiments performed in duplicate are shown. C. Same as in B, except cells were exposed to 1 μM Dox or Dox and IFN-γ for five hours. Percent of Annexin V-FITC stained positive cells compared to control is plotted on y-axis. Data are expressed as mean ± SD of three independent experiments performed in duplicate. Statistical significance was determined by comparing the difference between cells transfected with Lamin A/C siRNAs (12Asicon) and IRF5 siRNAs (12AsiIRF5) after each treatment; ** denotes P < 0.001. D. Cells were treated with the indicated doses of Dox or IR after siRNA transfection. Number of colonies is plotted on y-axis as percent of control. A total of 100% represents the number of colonies in control untreated 12Asicon cells. Data are expressed as mean ± SD of three independent experiments performed in duplicate. Statistical significance was determined by comparing the difference between colonies in 12Asicon versus 12AsiIRF5 cells after each treatment; * denotes P < 0.05.