Supplementary Figure 6: P-Cofilin levels change during mitotic exit and nuclear Cofilin-1 is essential for filament disassembly during mitotic exit.

(a) Representative immunostaining of p-Cofilin (grey, DAPI (blue)) in NIH3T3 cells treated with si-Control or si-Cofilin to validate specificity of the obtained fluorescence signals. Asterisks indicate presumably non-silenced cells. Scale bar, 10 μm. The experiment was performed once. (b) Images corresponding to quantifications shown in Fig. 6e. Confocal images show single slices at indicated time points after mitotic shake-off. Scale bar, 5 μm. (c) Immunoblot detecting p-Cofilin and Cofilin in RPE-1 cells after washout of nocodazole. Decreasing H3S10ph levels proof for successful release from the nocodazole-induced mitotic block. (d) P-Cofilin/Cofilin ratio was calculated by densitometric quantification of immunoblot intensities. Data are shown as mean + s.d. from n = 3 independent experiments. (e) Time-lapse imaging of NIH3T3 cells during mitotic exit corresponding to Fig. 6j. Cells stably express nAC-GFP (green) together with either WT- or NES-mCherry-Cofilin (red) and were treated with siRNA against the 3′-UTR of endogenous Cofilin-1. Scale bar, 10 μm. (f) Stably nAC-GFP expressing NIH3T3 cells were transfected with NLS-mCherry-Cofilin and followed during mitotic exit. Images show maximum intensity projections of confocal z-stacks and illustrate the absence of nuclear F-actin formation which was observed in 10 of 12 mitotic events. Time, hours:min; scale bar, 10 μm. (g) Immunoblot validating expression of opto-Cofilin in cells treated with either control siRNA or siRNA directed against the 3′-UTR of endogenous Cofilin-1. Unprocessed original scans of blots are shown in Supplementary Fig. 7.