Fig. 6: Nuclear-capture is required for perinuclear actin polymerization and nuclear G-actin depletion. | Nature Communications

Fig. 6: Nuclear-capture is required for perinuclear actin polymerization and nuclear G-actin depletion.

From: Nuclear-capture of endosomes depletes nuclear G-actin to promote SRF/MRTF activation and cancer cell invasion

Fig. 6

a H1299 cells were transfected with either EphA2-GFPWT (WT) or EphA2-GFPNLS2 (NLS2) (green) in combination with mCherry-Lifeact (red), with or without an siRNA targeting RhoG (siRhoG). Cells were challenged with HGF and monitored using time-lapse imaging (Supplementary Movies 4–6). Scale bar, 10 μm. Stills are presented in the inserts. Lifeact fluorescence intensity in the perinuclear region is expressed as a ratio between the perinuclear and the cortical regions. Solid and dotted lines are mean and SEM, respectively, n > 7 independent experiments, statistical test is two-way ANOVA and p-values are comparisons (at the indicated time points) between the EphA2-GFP WT + HGF values and other conditions as denoted by the colours. b Cells were incubated in the presence or absence of HGF for 20 min, fixed and stained with fluorescent DNaseI to visualize G-actin (G-actin; red) and counterstained with phalloidin (green) and DAPI (blue). The nucleus is denoted in red. Scale bar, 10 μm. c Cells were transfected with siRNAs targeting EphA2 (siEphA2) or control (nt), or siEphA2 in combination with wild-type EphA2-GFP (WT) or EphA2 NLS2-GFP (NLS2). Cells were challenged with (+HGF) or without HGF (−HGF) for 20 min. Nuclear G-actin content was quantified by flow cytometry. Box and whisker plots (min. to max. whiskers, + represents mean) are ratio of the geometrical mean value of HGF-treated vs. non-treated cells (+HGF/−HGF), n = 7 (control, left panel), n = 3 (siEphA2, left panel), and n = 5 (right panel) independent experiments, statistical test is unpaired t-test two-sided analysis. d Cells expressing MRTF-GFP were transfected with siRNAs targeting EphA2, RhoG or R62D, or nuclear-targeted R62D actin mutants or vector control, and challenged with HGF as indicated. Distribution of MRTF-GFP was expressed as the ratio of MRTF-GFP in the nucleus vs. the cytoplasm. Values are expressed as mean ± SEM, n = 3 independent experiments, statistical test is two-way ANOVA. e Cells were transfected with R62D actin mutant or vector control. Scattering was determined as for Fig. 2c. Box and whiskers: 10–90 percentile whiskers, + represents mean, black line represents median, n = 3 independent experiments, statistical test is one-way analysis ANOVA.

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