Supplementary Figure 2: Response of ATM RQ mutant to oxidative and DNA damage.
From: ATM functions at the peroxisome to induce pexophagy in response to ROS
(a) Subcellular fractionation of Zellweger (GM13267) fibroblasts demonstrating the localization of phospho-ATM (S1981) and ATM in response to H2O2 (0.4 mM, 1 h) in the indicated subcellular compartments. LDH, Lamin A/C and β-integrin were used as markers for cytosol (C), nuclear (N) and membrane (M) fractions, respectively. WCE, whole cell extract. (b) AT (GM05849) fibroblasts were transfected with either wild type (WT) or peroxisome localization-deficient R3047Q (RQ) mutant Flag-tagged ATM and treated with H2O2 (0.4 mM) for 1 h. ATM was immunoprecipitated using anti-Flag antibody and activation determined using phosphoserine S1981 specific antibody. (c and d) ATM kinase assay with MR complex, Nbs1, and DNA were performed with 0.2 nM dimeric ATM, 50 nM GST-p53 substrate, 96 nM MR, 195 nM Nbs1, and 140 nM linear double stranded DNA. Phosphorylation of ATM was detected by SDS-PAGE and western blotting with an antibody to p53 phospho-Ser15. (e) Frequency of γ-H2AX foci in AT cells (AT5, GM05849) and HepG2 cells + siATM at different time points post irradiation of 2 Gy. 100 cells were analyzed for each time point in each experiment (Mean ± s.d., n = 3 independent experiments, student’s t test). The mean number of foci is plotted against time. ∗P < 0.05, ∗∗P < 0.01. (f) Representative figures of γ-H2AX foci. A: Untreated cells; B: AT5 (GM05849) + ATM cells 30 min post irradiation; C: AT5 cells 30 min post irradiation; D: AT5 cells 180 min post irradiation; E: AT5 + ATM cells 180 min post irradiation; F: AT5 + ATM-R3047Q cells 180 min post irradiation. Scale bar, 10 μm. (g) Representative images of chromosome aberrations at metaphase. A: AT5 + ATM control metaphase; B: AT5 + ATM showing dicentric (big arrow) and breaks (small arrow); C: AT5 showing radials (big arrow) and breaks (small arrow); D: AT5 + ATM-R3047Q showing radials (big arrow) and breaks (small arrow). (h) Chromosome aberrations at metaphase post irradiation of 3 Gy in AT cells (AT5, GM05849) and HepG2 cells + siATM. For each group 50 metaphases were scored in each experiment (mean ± s.d., n = 3 independent experiments, student’s t test). ∗∗P < 0.01. Uncropped images of western blots are shown in Supplementary Fig. 9. Statistical source data for Supplementary Fig. 1e, h can be found in Supplementary Table 1.
