Research Paper Volume 6, Issue 4 pp 248—263

Figure 8. Ionizing radiation induces altered nuclear staining of methylated TRF2 in a ATM-dependent manner. (A) Ionizing radiation induces cellular senescence in GM9503 cells. GM9503 (p23) cells were treated with 12 Gy IR. Senescence-associated β-galactosidase assays were performed 48 h post IR. (B) Indirect immunofluorescence with anti-TRF2-2meR17 antibody in mock- or IR-treated GM9503 cells (p23). Cell nuclei were stained with DAPI in blue. (C) Quantification of percentage of cells with altered nuclear staining of methylated TRF2. A total of 1000 cells in triplicate were scored in blind for untreated or IR-treated cells fixed at various time points post IR as indicated. Standard deviations from three independent experiments are indicated. (D) Western analysis of GM9503 cells (p23) that were either mock- or IR-treated. Immunoblotting was performed with anti-TRF2-2meR17 or anti-TRF2 antibody. The γ-tubulin blot was used as a loading control. (E) ATM inhibition abrogates IR-induced altered nuclear staining of methylated TRF2. GM9503 cells (p23) were treated with DMSO or KU55933 prior to 12 Gy IR treatment. Forty-eight hours post IR, cells were processed for indirect immunofluorescence with anti-TRF2-2meR17 antibody. Quantification of percentage of cells with altered nuclear staining of methylated TRF2. A total of 1000 cells in triplicate were scored in blind. Standard deviations from three independent experiments are indicated. (F) Little IR-induced altered nuclear staining of methylated TRF2 is observed in AT2RO cells lacking functional ATM. Quantification of percentage of cells with altered nuclear staining of methylated TRF2. A total of 1000 cells in triplicate were scored in blind. Standard deviations from three independent experiments are indicated. (G) Sequential extraction of the nuclear matrix from GM9503 cells treated with either DMSO or KU55933. Immunoblotting was performed with anti-TRF2-2meR17, anti-TRF2 or anti-Lamin A antibody. (H) Sequential extraction of the nuclear matrix from AT2RO cells that were either untreated or treated with 12 Gy IR. Immunoblotting was performed with anti-TRF2-2meR17, anti-TRF2 or anti-Lamin A antibody.