Extended Data Fig. 4: Plasma membrane V-ATPase feeds into Rab7-positive endosomes.
From: Plasma membrane V-ATPase controls oncogenic RAS-induced macropinocytosis
a, b, Fluorescence micrographs of HeLa control and HRAS(G12V) cells with the indicated treatment showing V1A (green) and Rab7 (red) immunostaining. Representative original image used to calculate Mander’s overlap coefficient (a) and processed image (b). c, d, Quantification of Rab7 with V1A colocalization in HeLa cells treated as indicated using Mander’s overlap coefficient (M coefficient). e, f, Epifluorescence imaging of self-complementing GFP from HeLa HRAS(G12V) cells transfected with V1A–GFP11 and plasma membrane-targeting Lyn–GFP(1–10). Positive fluorescence indicates V1A localization to the plasma membrane. e, Fluorescence micrographs of time-lapse imaging. The boxed area of the cell (left) was enlarged (four right images) to show plasma membrane V-ATPase being internalized over time and forming a vesicle (arrow). Time (in s) is shown on each micrograph. f, Fluorescence micrographs of self-complementing GFP (left), immunofluorescence of Rab7 (middle) and merge of V1A and Rab7 (right) with boxed area enlarged beneath the image to show Rab7 colocalization with plasma membrane-derived V1A. In a, b, images are representative of three biological replicates. For c, d, 25 cells were quantified in each biological replicate (n = 3); data are mean ± s.e.m.; unpaired two-tailed Student’s t-test. Scale bars, 10 μm.
