Fig. 1: HGF promotes nuclear-capture of EphA2-positive endosomes. | Nature Communications

Fig. 1: HGF promotes nuclear-capture of EphA2-positive endosomes.

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

Fig. 1

a H1299 cells were surface-biotinylated and allowed to internalize in the presence or absence of HGF. Biotin remaining at the cell surface was removed and distribution of biotinylated proteins visualized with streptavidin (green) and counterstained with DAPI (blue) and LaminA/C (red). Scale bar, 10 μm. Internalized particles (lower graph) and their distance from the nuclear surface (upper graph) was quantified. Values are mean ± SEM (upper graph) (n = 3 individual experiments), statistical significance was determined by one-way analysis of variance (ANOVA). Box and whiskers: 10–90 percentile whiskers, + represents mean, black line represents median (lower graph). b, c SILAC-labelled cells were surface-biotinylated and internalized as for a. Nuclei were purified and the biotinylated proteome determined by mass spectrometry (schematic). Western blotting confirmed integrity of the nuclear preparations (c). The scatter plot indicates the SILAC ratios of the biotinylated nuclear-associated proteome (+HGF/−HGF) from two independent experiments ((1) and (2)) plotted on the x and y-axes, respectively. Biotinylated proteins enriched in the nuclear fraction in response to HGF are represented in the upper right-hand quadrant. Proteins moving from the plasma membrane to the nuclear fraction are highlighted by red dots. d Surface-biotinylated cells were allowed to internalize in the absence or presence of HGF for the indicated times. Nuclei were purified and the presence of biotinylated-EphA2 in these purified nuclei determined using a capture-ELISA (left graph) and by super-resolution microscopy (right panels, scale bar 5 μm). Data are expressed as the proportion of surface-labelled EphA2, which is translocated to the nuclear preparation. Values are mean ± sem, n = 3 individual experiments, statistical test is repeated-measures two-way ANOVA. e H1299 cells were transfected with siRNAs targeting EPHA2 (siEphA2) or a control (nt), surface-labelled and allowed to internalize in the presence or absence of HGF. Internalized biotinylated particles were determined as for a. Values are mean ± SEM (left graph) (n = 3 individual experiments), statistical significance was determined by two-way analysis of variance (ANOVA). Box and whiskers: 10–90 percentile whiskers, + represents mean, black line represents median (right graph).

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