FIG. 5.

Cells expressing the ATR phosphorylation site mutants arrest in G₂ following release from replication arrest. (A) Western blotting of crude nuclear extracts to show equivalent BLM expression in control PSNF5 cells (lane 1) and four independent clones of GMO8505 cells expressing BLM-T99A/T122A is shown; clone C2.1 (lane 3), clone C20 (lane 4), clone C1.2 (lane 5) and clone C1.4 (lane 6) are depicted. Lane 2 indicates the untransfected GMO8505 cells for validation of the antibody. β-Tubulin was used as loading control. (B) Exponentially growing cultures of either PSNF5 cells expressing WT BLM or clone 1.4 cells expressing BLM-T99A/T122A were exposed to HU (2 mM) for 16 h and then released into drug-free medium. The DNA content was analyzed by flow cytometry at the times indicated. (C) Analysis of a caffeine-sensitive G₂/M checkpoint in HU-treated cells expressing BLM-T99A/T122A. Cells were treated with HU as before and then released in the presence or absence of 4 mM caffeine. Cells were harvested at the time points indicated and analyzed by flow cytometry for expression of phosphorylated histone H3 to determine the percentage of cells in mitosis (left panel) and for DNA content (right panel). Cells expressing phosphorylated histone H3, and with a 2 N DNA content, are circled and labeled with an M for the 24-h time point. The percentage of cells expressing phosphohistone H3 is shown graphically. Results are expressed as the means of three experiments. Error bars indicate standard errors of the means. (D) Expression of phospho-Chk1. Whole-cell extracts were prepared from cells expressing T99A/T122A-BLM at the times indicated after release from HU block. The extracts were analyzed by Western blotting for total Chk1, phospho-Chk1 (Ser 345), and β-tubulin levels (shown by representative blots). The bar graph indicates quantification of the blotting data (normalized for β-tubulin levels) and represents the means of two determinations.