Figure 3.

Different dynamics of PARP1-GFP and mCherry-PARP1 after staurosporine exposure.A, schematic diagram of PARP1-GFP and mCherry-PARP1 constructs. B, PARP1-GFP and mCherry-PARP1 expression in HeLa cells. After 1-day transfection, Western blotting using anti-PARP1 antibody was performed to confirm expression. The lower arrow indicates endogenous PARP1 expression in HeLa cells, whereas the upper arrow shows exogenous PARP1 expression. C, localization of PARP1 constructs after 6-h exposure to staurosporine (300 nM). Nuclei were stained with DAPI (blue). Yellow lines indicate the position of nuclei. The scale bar represents 10 μm. D and F, effect of caspase activation on the recruitment of PARP1 constructs to DNA lesions induced by microirradiation. zVAD-fmk (50 μM) was added for 30 min before 2-h exposure to staurosporine (300 nM). Cells transiently transfected with PARP1 constructs were subjected to microirradiation at the indicated circle. The scale bar represents 10 μm. Right graphs indicate the time course of fluorescence of PARP1 constructs at circles. Images were taken every 2 s for 10 min. E and G, effect of caspase activation on the mean amplitude (left) and decay time constant (right) of PARP1 constructs after microirradiation without or with staurosporine (300 nM) and zVAD-fmk (50 μM) (means ± S.E.M., n = 8). ∗∗p < 0.01, ∗∗∗p < 0.001 versus control (one-way ANOVA with post hoc Tukey test). DAPI, 4',6-diamidino-2-phenylindole; PARP1, poly(ADP-ribose) polymerase 1.