Figure 2.

Effect of UTX knockdown H3K27me3 levels and tumor cell radiosensitivity. A) Expression of H3K27me3 demethylases (UTX and JMJD3) and H3K27me3 in the 3 tumor cell lines and two normal lung fibroblast cell lines (MRC5 and MRC9). Protein from whole cell lysates (20μg for UTX and 60μg for JMJD3), and chromatin protein (2μg) were subjected to immunoblot analysis with actin and H3 used as loading controls, respectively. B) Tumor cells were transfected with nontargeted (nt) siRNA or siRNA specific for UTX (UTX siRNA) and 48h post-transfection whole cell lysates was subjected to immunoblot analysis with actin used as a loading control. C) Transfected tumor cells were irradiated with 10Gy (IR) and collected at 1h later for immunoblot analysis of H3K27me3 with H3 used as a loading control. Representative immunoblots of 2 independent experiments are shown; ratios of H3K27me3 in irradiated versus unirradiated are shown below each blot. D) 48h after transfection U251, MDA-MB-231, and A549 cells were plated, irradiated after attachment and subjected to clonogenic survival analysis. Colony-forming efficiency was determined 10 to 14 days later and survival curves were generated after normalizing for cell killing from siRNA alone. DEFs were calculated at a surviving fraction of 0.1. Values shown represent the mean ± SEM for 3 to 4 independent experiments. *p≤0.04 according to Student’s t test.