Extended Data Fig. 5: sAC–PKA pathway is necessary for oncogenic RAS-induced macropinocytosis.
From: Plasma membrane V-ATPase controls oncogenic RAS-induced macropinocytosis
a–g, Effect of sAC (KH7) or PKA (H89) inhibition on membrane cholesterol, RAC1 activation and RAC1 localization. a, Fluorescence micrographs of HeLa HRAS(G12V) cells in indicated treatments with filipin labelling (top), membrane labelling with R-pre (middle), and merge of filipin and R-pre fluorescence micrographs with boxed areas enlarged beneath the image to show plasma membrane localization (bottom). b, Quantification of the ratio of filipin to R-pre membrane localization with the indicated treatments in HeLa HRAS(G12V) cells. c, Immunoblot of endogenous RAC1 activity (GST–PBD, pull-down of RAC1–GTP; tubulin loading control) in HeLa control and HRAS(G12V) cells treated as indicated. d, Immunoblot of endogenous RAC1 in the plasma membrane fraction and whole-cell lysate in HeLa control and HRAS(G12V) cells treated as indicated. e, Fluorescence micrographs of cholesterol (filipin) distribution in HeLa control cells with indicated treatments. f, Quantification of plasma membrane cholesterol in HeLa control cells with the indicated treatments. g, Fluorescence micrographs of cholesterol (filipin) distribution in BxPC-3 cells in the absence or presence of ectopically expressed KRAS(G12V) treated as indicated. g, Data are mean ± s.e.m. representing the fraction of cells that display cholesterol plasma membrane localization. Images (a, e, g) and immunoblots (c, d) are representative of three biological replicates. Scale bars, 10 μm. At least 20 (b), 50 (f) and 500 (g) cells were quantified in each biological replicate (n = 3); data are mean ± s.e.m.; unpaired two-tailed Student’s t-test. Gel source data for c, d are shown in Supplementary Fig. 1.
