Figure 4

Inhibition of TMZ-induced G2 arrest by Rsv leads to mitotic catastrophe. U87 cells were treated with Rsv 30 μM, TMZ 100 μM or RT for 24, 38 and 48 h, followed by fixation and DAPI staining. (A) representative images of nuclei from cells treated with DMSO (control), Rsv for 38 h or RT for 48 h. Double arrows point to fragmented/irregular nuclei; single arrow points to micronuclei; double arrowheads point to enlarged nuclei; Scale bar: 6 μm; (B) representative visible and fluorescent images of nuclei from untreated cells showing a normal mitosis (i and ii); an abnormal chromosome condensation and mitosis, with cellular enlargement, in a cell treated with RT for 48 h (iii and iv); and a triple mitosis in a cell treated with Rsv for 48 h (v and vi) and. Scale bar: 10 μm; (C) direct counting of the percentage of nuclei presenting normal (top), irregular (mid) or large phenotype (bottom); *p<0.05, **p<0.01 and ***p<0.001 in relation to control; (D) DAPI-stained nuclei were analyzed for size and irregularity using the NMA tool, as described on material and methods, and the percentage of each nuclear type is shown. At least 200 nuclei in three independent experiments were analyzed in each data point; *p<0.05 and **p<0.01 in relation to control; ^#p<0.05 and ^##p<0.01 in relation to TMZ and Rsv alone.