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. Author manuscript; available in PMC: 2011 May 18.
Published in final edited form as: Cell Cycle. 2010 Apr 15;9(8):1617–1628. doi: 10.4161/cc.9.8.11470

Figure 5. ICRF-193 induces ATM-dependent checkpoint signaling, but not DNA damage in human fibroblasts and lymphoblasts.

Figure 5

A. Normal and AT lymphoblasts were treated with colcemid and DMSO or ICRF-193 for 3 or 6 h and harvested for western immuno-blot analysis. Equal amounts of cell lysate protein were analyzed for phospho-Ser 345 CHEK1, phospho-Thr 68 CHEK2, and phospho-Ser 15 p53. For γH2AXdetection, equal numbers of cells were boiled in gel loading buffer before SDS-PAGE analysis. ICRF-193 treatment induced ATM-dependent phosphorylation of Thr 68-Chk2 and Ser 15-p53, but not γH2AX. A small increment of phophorylated Ser345-Chk1 is observed in the normal lymphoblasts upon 6 hr of ICRF-193 treatment, suggesting that lymphoblasts may activate low levels of Chk1 after a prolonged exposure to ICRF-193. Normal lymphoblasts treated with 12 μM etoposide for 6 h were analyzed as a positive control for DNA damage-induced signaling responses. B. Normal and AT fibroblast lines were treated with DMSO or 4 μM ICRF-193 for 3 h and harvested for western immunoblot analysis. Levels of γH2AX and phospho-Ser345 Chk1 remained low in all fibroblast lines examined. Similar to the normal lymphoblasts described in Fig. 1A, an ATM-dependent phosphorylation of Thr 68-Chk2 and Ser15-p53 were detected upon treatment with ICRF-193. NHF1hTERTs treated with 12 μM etoposide for 6 h were analyzed as a positive control for DNA damage.