Extended Data Fig. 1: V-ATPase is required for RAS-induced macropinocytosis.
From: Plasma membrane V-ATPase controls oncogenic RAS-induced macropinocytosis
a, Functional clusters within the macropinocytosis screen hits defined by STRING analysis (pink, primary screen; red, primary and confirmation screen). b, Quantification of TMR–dextran uptake following knockdown of the indicated V-ATPase subunits in HeLa HRAS(G12V) cells (HV12). c, Immunoblot of V1A expression from whole-cell lysate (vinculin loading control). d, Effect of V-ATPase depletion (siV1A) on cholesterol localization in Hela HRAS(G12V) cells. Fluorescence micrographs of filipin staining (left), membrane labelling with R-pre (a transfected construct containing a modified sequence of the membrane targeting domain of KRAS linked to RFP, middle), merge of filipin and R-pre with boxed areas enlarged to show plasma membrane localization (right), and the quantification of the ratio of filipin to R-pre membrane localization (bar graph) in control (siCtl) or V1A-knockdown conditions. e, Effect of V1A knockdown on total cholesterol in HeLa HRAS(G12V) (left) and KRAS(G12V) (KV12) (right) cells. f, Effect of bafilomycin A1 (BafA1) and rescue by exogenous cholesterol on the localization of cholesterol and RAC1 in HeLa HRAS(G12V) cells. Fluorescence micrographs of filipin (top), GFP–RAC1 (bottom) and quantification of relative surface GFP–RAC1 (bar graph). g, Effect of oncogenic RAS and V1A expression on RAC1 localization. Immunoblots of RAC1 and V1A in the plasma membrane fraction and whole-cell lysate from HeLa T7-vector control (Ctl) and HRAS(G12V) or oncogenic KRAS cell lines with or without V1A knockdown. Images (d, f) and immunoblots (c, g) are representative of three biological replicates. In f, the dashed lines delineate the cell and nucleus and data (mean ± s.e.m.) represent the fraction of cells that display plasma membrane localization of cholesterol. Scale bars, 10 μm. At least 500 (b, f) and 20 (d) cells were quantified in biological replicates (n = 3). In e, cholesterol quantification is representative of four biological replicates. All data are mean ± s.e.m. for the indicated sample size; unpaired two-tailed Student’s t-test. Gel source data for c, g are shown in Supplementary Fig. 1.
