Figure 4.
Targeting the HBP pathway via silencing of either OGT or OGA alters DSB repair, IRIF resolution, viability and IR-induced senescence in MCF7TagRFP-IBD cells. A, Silencing of OGT or OGA has opposing effects on IRIF resolution in MCF7 cells. TagRFP-IBD foci in representative cells 24 h after 6 Gy are shown. Scale bar 20 μm. B, Plots of IRIF per nucleus in individual cells. Red bar indicates mean ± SEM; *, P <0.05; ***, P < 0.0001, (Mann-Whitney test). C, Effects of OGT or OGA silencing on DSB repair. Cell lines with inducible shRNA-miRs targeting OGT, OGA or with scrambled control were grown in medium containing different concentrations of glucose for 24 h. Following irradiation with 6 Gy, neutral comet assays were performed 24 h post IR. Plots of percent tail DNA are shown. Red line indicates mean ± SEM. ***, P < 0.0001, (Mann-Whitney test). D, Immunofluorescence staining of endogenous 53BP1 and γH2AX in shScr control, shOGA, and shOGT cells 24 h after 6 Gy. Shown are individual channels and overlay of anti-γH2AX (red), anti-53BP1 (yellow), shRNA-miR expression (green), and DAPI (blue) for representative cells. Mean γH2AX foci per cell ± SEM is indicated. Below are perspective plots of γH2AX staining intensity. Scale bar 20 μm. E, Clonogenic survival of cells after irradiation. Normalized survival fractions indicating average of 3 replicates are shown. F, Senescence induction in cells bearing shRNA-miRs targeting OGT or OGA evaluated by SA-β-Gal (blue) staining 5 days after 6 Gy. Representative images are shown. Percent SA-β-Gal positive cells are indicated as mean ± SEM. Scale bar 50 μm.