Figure 8.
Expression of phospho-mimic CHK1 rescues sensitivity to Aph and replication fork slowing in WS and WSWRN6A cells. (A) WS and WSWRN6A cells were transfected with Flag-tagged CHK1 S317D/S345D mutant (CHK1317/345D). Lysates were collected 48 h thereafter and expression levels of Flag-CHK1317/345D were determined by immunoblotting with anti-Flag antibody. WSWRN cells were used as negative control. Total WRN was used to assess the amount of wild-type or mutant form of WRN. Lamin B1 was used as loading control. (B) Effect of the expression of the Flag-CHK1317/345D plasmid on chromosomal damage. After transfection, cells were treated or not with Aph for 24 h. The graph shows the average overall chromosome gaps and breaks. Data are presented as means of three independent experiments. Error bars represent standard error on the mean. (C) Evaluation of replication fork recovery in WS and WSWRN6A cells transfected with Flag-CHK1317/345D. Cells were treated as indicated in the experimental scheme. Aph (0.4 μM) and roscovitine (100 μM) were added for the indicated time. Median values are represented as horizontal black lines. (D) Percentage of new origin firing in cells treated as in (C). Data are reported as mean from three independent experiments. Error bars represent standard error. **** = Statistically significant P < 0.0001 (Student's t-test).