BLM Deficiency Is Not Associated with Sensitivity to
Hydroxyurea-Induced Replication Stress
Figure 2
Levels of HU-induced DSBs are similar in BLM-deficient cells and control cells. (a) HeLaV and HeLashBLM cells were transfected as in Figure 1(a). Upper panel: After 24 h, cells were left untreated (0) or were treated with 2 mM HU for the indicated times. Protein extracts were subjected to SDS-PAGE. The membrane was probed with anti-BLM (C18), anti-Chk1 pS345, anti-Chk2 pT68, and anti--H2AX antibodies, and with anti--actin antibody, as a loading control. The membrane was stripped and reblocked between successive antibody incubations. Lower panel: After 24 h, cells left untreated or treated with 2 mM HU for 16 h were harvested, fixed, and analyzed by FACS. (b) BS-GFP and BS-GFP-BLM cells were left untreated or were treated with 2 mM HU for the indicated times. Protein extracts were subjected to SDS-PAGE and the bands were transferred to a membrane. The membrane was probed with anti-BLM (C18), anti--H2AX antibodies, and with anti-GAPDH antibody, as a loading control. Lower panel: After 24 h, cells left untreated or treated with 2 mM HU for 16 h were harvested, fixed, and analyzed by FACS. (c) Representative images of HeLa cells in the comet assays. (Left) Untreated cell. (Right) Example of cell displaying an increase in DNA migration after 48 h of HU treatment, due to DNA breaks. (d) The effect of 2 mM HU treatment on DNA migration (tail moment) in the comet assays with HeLaV and HeLash BLM cells transfected as in Figure 1(a). After 24 h of transfection, cells were left untreated or were treated with 2 mM HU for 16 h or 48 h. The cells were subjected to comet assays (see Section 2). Two independent experiments were analyzed for each set of conditions. Between 100, and 110 cells were scored for each set of conditions, in each experiment. Error bars represent standard errors of the mean. (e) As in (d), using BS-GFP-BLM or BS-GFP cells left untreated or treated with 2 mM HU for 16 h or 48 h.
