sublines, Q3SA and Q3SC, as contrasting examples without or with ATM expression, respectively. ATM activity in sample iPSC lines was confirmed by the X-irradiation (XR)-induced phosphorylation of CHK2 as detected by western blots (Figure 1E). The pCHK2 band was enhanced by XR (8 Gy) and diminished by pre-treatment with the ATM-specific inhibitor KU-55933 (KU) in unrelated control iPSC (SC1) and in Q3SC. Little pCHK2 was detected in Q1SA. Results were confirmed by observing the presence of XR-induced γH2A.X nuclear puncta with the same cell lines exhibiting positive (CAR3 and Q3SC) or negative (Q1SA and Q3SA) results (Figures 1F and S1C). Therefore, Q1SA expressed full-length ATM protein but had little or no kinase activity, presumably because of the presence of a previously unreported C > T variant at position c.7181, producing a S2394L that apparently affects activation. However, results indicate both the presence of ATM protein and its function in the Q3SC subline where none was expected on the basis of the diagnostic genotype provided with the subject cells. Results from Q3SA matched those expectations, as no ATM protein or activity was observed, with one allele terminating shortly after a frameshift mutation (c.217_218 delGA) and the other allele terminating at a
