Fig. 7.

Analyses of p53-KO and TRI-KO fibroblasts for cell cycle regulation, DNA damage checkpoints, and apoptosis, after a genotoxic treatment. The indicated cell lines were irradiated and probed for various damage response endpoints. (A) Wee-1 expression is up-regulated in triple knockout fibroblasts, which is in agreement with previously published data (15), and this level is not affected by UV. (B) Both the ATR → Chk1 and ATM → Chk2 checkpoint pathways appear to be functioning normally in the absence of cryptochrome, as evidence by phosphorylation of signal transducing kinases after UV treatment. Dose-response (2 h time point after 0, 2.5, 5, and 10 J/m² of UV treatment) and kinetic experiments (1, 2, and 4 h after 5 J/m² of UV treatment) did not reveal any difference in Chk1 and Chk2 phosphorylation in fibroblasts of the 2 genotypes (data not shown). (C, D) Assay for UV-induced apoptosis. The p53-KO and TRI-KO fibroblasts were irradiated with UV and tested for apoptosis by caspase 3 cleavage (24 h) and PARP cleavage, 24 h after UV dose treatments at 0, 10, 20, and 50 J/m². Cell lysates were also probed for actin as a loading control.