Extended Data Fig. 7: The SLC4 family is required for PKA-dependent, RAS-induced macropinocytosis.
From: Plasma membrane V-ATPase controls oncogenic RAS-induced macropinocytosis
a, Quantification of TMR–dextran uptake in the absence (bicarbonate-free) or presence (bicarbonate) of extracellular bicarbonate in mutant RAS cells. b, Fluorescence micrographs of V1A immunostaining following treatment of mutant RAS cells with vehicle or pan-SLC4 inhibitor (S0859). c, Fluorescence micrographs of HeLa HRAS(G12V) cells in indicated treatments with V1A immunostaining (top), membrane labelling with R-pre (middle), and merge of V1A and R-pre fluorescence micrographs with boxed areas enlarged beneath the image to show plasma membrane localization (bottom). d, Quantification of the ratio of V1A to R-pre membrane localization with the indicated treatments in HeLa HRAS(G12V) cells from c. e, f, Effect of SLC4 inhibition (S0859) and rescue (S0859 + CA-PKA) in HeLa HRAS(G12V) cells. e, Fluorescence micrographs of V1A immunostaining and filipin labelling following indicated treatments. Dashed lines delineate the cell and nucleus. f, Quantification of TMR–dextran uptake. g, Fluorescence micrographs of cholesterol (filipin) distribution in BxPC-3 cells in the absence or presence of ectopically expressed KRAS(G12V) treated with SLC4 family inhibitor (S0859). h, mRNA levels of SLC4A7 expression in HeLa control, HRAS(G12V) or KRAS(G12V) cells. i, mRNA levels of SLC4A7 expression following KRAS knockdown in MIA-PaCa-2 cells. j, Effect of PI3K (LY294002, left) or MEK (U0126, right) inhibition on SLC4A7 expression in HeLa control and HRAS(G12V) cells. Immunoblots of SLC4A7 expression from whole-cell lysate (vinculin loading control). p-AKT (left) and p-ERK (right) immunoblots show inhibition of pathways by the indicated treatments. k, Effect of PKA (H89), NHE (EIPA), SLC4A7 (siSLC4A7) and V1A (siV1A) inhibition on submembranous pH (pHsm) in HeLa HRAS(G12V) cells transfected with SEpHluorin–mCherry construct (genetically encoded ratiometric pH probe that is targeted to the inner leaflet of the plasma membrane). Calibration curve of SEpHluorin–mCherry (line graph) was performed with K+ nigericin buffer. Quantification of submembranous pH with H89 and EIPA treatment (bar graph, middle) or with knockdown of SLC4A7 and V1A (bar graph, right). l, Immunoblot of SLC4A7 (vinculin loading control) in MIA-PaCa2 cells with doxycycline-inducible SLC4A7 depletion. m, n, Effect of doxycycline-inducible SLC4A7 depletion in BxPC-3 cells on tumour growth. m, Immunoblot of SLC4A7 expression from whoe-cell lysate (vinculin loading control). n, Waterfall plots of xenografts treated as shown relative to baseline. Each bar represents a tumour. Images (b, c, e, g), immunoblots (j, l, m), and mRNA levels (h, i) are representative of three biological replicates. b, e, g, Data are mean ± s.e.m. representing the fraction of cells that display V1A (b, e) or cholesterol (g) plasma membrane localization. Scale bars, 10 μm. At least 20 (d) and 500 (a, b, e–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 j, l, m are shown in Supplementary Fig. 1.
