Figure 1.

NEU causes activation of checkpoint signalling pathways in a dose dependent manner. (A) MCF7 cells were treated with 0, 2, 6 and 10 mM NEU for 2 hours and lysates were analysed for activation of checkpoint proteins by immunoblotting. (B) MCF7 cells were treated with 0, 0.3, 0.6, 1.2 and 1.8 mM NEU for 2 hours and lysates were analysed for activation of checkpoint proteins by immunoblotting. (C) HeLa cells were treated with 0.3 and 10 mM NEU for 1 hour, fixed and analysed for γH2AX foci formation by immunostaining. DMSO was used as negative control and neocarzinostatin (NCS), an IR-mimetic drug, was used as positive control at a concentration of 200 ng/ml. Scale bar: 20 μM. (D) HeLa cells were treated with 0.3 and 10 mM NEU for 2 hours, fixed and analysed for pRPA foci formation by immunostaining. DMSO was used as negative control. Scale bar: 20 μM. (E) HeLa cells were treated with 0, 0.3 and 10 mM NEU for 2 hours and lysates were analysed for phosphorylation of RPA. (F and G) MCF7 cells treated with 0 and 10 mM NEU for 2 hours were collected, embedded in agarose and layered on slides. Cells were subjected to lysis followed by electrophoresis in neutral and alkaline conditions respectively. N = 150 cells (50 cells/experiment). Data shown are mean +/− standard error. The results for % DNA in tail and tail length are significantly different at p < 0.05 in Mann Whitney U test and student’s t test respectively.