SUPPLEMENTAL FIGURE LEGENDS. ... Cells were fixed 15 minutes after 5 Gy of gamma ... was assessed by immunofluorescence with anti phosphorylated histone H2AX antibody.
SUPPLEMENTAL FIGURE LEGENDS
Figure S1. Specificity of anti-ATM-S1981P antibody. A. SV40 transformed human fibroblast cells, GM637 (ATM +/-) and AT9607 (ATM-/-) were exposed to 1 Gy of ionizing radiation or 20 J/m2 of ultraviolet (UV) irradiation and harvested fifteen minutes or 3 hours later, respectively. Total and phosphorylated ATM, SMC1 and NBS1 were assessed by immunoblots of whole cell lysates. Phosphorylated histone H2AX was immunoblotted after acid cellular extraction. B. Subcellular localization of phosphorylated ATM, H2AX and SMC1 in GM637 cells and AT9607 cells. Cells were fixed 15 minutes after 5 Gy of gamma irradiation (IR) or 3 hours after 20 J/m2 of UV irradiation (UV) and then analyzed by immunofluorescence with the indicated antibodies.
Figure S2. Subcellular localization of phosphorylated p53 after IR or chloroquine treatment. MCF7 cells were exposed to 1 Gy of ionizing irradiation and fixed 15 minutes later, or treated with 40 (g/mL of chloroquine for one hour and then fixed. Subcellular localization of phosphorylated p53 at serine 15 was analyzed by immunofluorescence with phospho-specific antibody (p53S15p). Presence of double strand breaks was assessed by immunofluorescence with anti phosphorylated histone H2AX antibody. Nuclei were visualized by staining with DAPI.
Figure S3. Cell cycle dependencies of focus formation with phosphorylated ATM, BRCA1, and phosphorylated SMC1. A. Human primary fibroblast cells (1070SK) were incubated in serum-reduced media for two weeks (G0), or exponentially grown (cycling). Cell cycle distribution was analyzed by flow cytometry of propidium iodide (PI) stained cells. Presence or absence of cells in S phase was assessed by bromodeoxyuridine (BrdU) incorporation after incubation in BrdU-containing media for 8 hours. BrdU labeled cells were visualized by immunofluorescence with FITC-conjugated anti BrdU antibody (shown in green). Cells in each culture were exposed to 0 Gy (IR-) or 1 Gy (IR +) of gamma irradiation and fixed 15 minutes later. Subcellular localization of phosphorylated ATM, BRCA1 and phosphorylated SMC1 was assessed by immunofluorescence with indicated antibodies. B. 1070SK cells arrested in G0 by serum starvation (G0) or exponentially growing cells (cycling) were exposed to 0 Gy (IR -) or 1 Gy (IR +) of gamma irradiation and harvested 15 minutes later. Total BRCA1 and total and phosphorylated ATM and SMC1 were assessed by immunoblots with indicated antibodies. C. HCC1937 (BRCA1-deficient) cells were transiently transfected with human expression vector carrying [BRCA1wt] or not carrying [vec] a wild-type BRCA1 gene. Twenty-four hours after transfection, cells were treated with 0.5 (M of TN-16 for 10 hours. The cells blocked at mitosis were collected by selective detachment, then released by washing with fresh media twice. Synchronized HeLa cell culture was prepared in the same manner. Cell cycle progression was monitored by cell cycle distribution at indicated times after release from TN-16 arrest by flow cytometry analysis of PI stained cells. Cells were irradiated with 0 Gy (IR-) or 5 Gy (IR+) of gamma irradiation at indicated times after release from TN-16 arrest and fixed 15 minutes later. Subcellular localization of phosphorylated ATM and BRCA1 were analyzed by immunofluorescence with indicated antibodies.