Extended Data Fig. 4 |. Macropinocytosis enables proliferation of KRAS-mutant cells under conditions where aspartate is limited.

(a) Fold change in cell number (log₂) of HeLa (left) or BxPC-3 tet-KRASV12 (right) cells cultured under indicated concentrations of oxygen for 5-6 days in the presence or absence of 1% BSA. BxPC-3 tet-KRASV12 cells were cultured in the presence or absence of doxycycline (0.1μ/mL) to activate KRASV12 expression. (b) Immunoblot analysis of several members of the ETC complex in parental MIA PaCa-2 cells or Rho(0) counterparts. ACTB was used as a loading control. (c) Representative bright-field micrographs of HY15549 cells cultured for 5 days in media with or without 2% BSA. Where indicated, cells were treated with bafilomycin A1 (BafA1, 10 nM), ferric ammonium citrate (FAC, 0.1 μg/mL) and complex III inhibitor antimycin A (Anti. A, 100 nM). Scale bar = 50 μm. (d) Immunoblot analysis of GOT1 and GOT2 in GOT1/2- double knockout BxPC-3 tet-KRASV12 cells compared to parental controls. ACTB was used as a loading control. (e) Fold change in cell number (log₂) of GOT1/2- double knockout BxPC-3 tet-KRASV12 cells cultured in the indicated media conditions for 7 days in the presence or absence of doxycycline (0.1 μg/mL) (top). Representative bright-field micrographs of GOT1/2- double knockout BxPC-3 tet-KRASV12 cells in media supplemented with 1% BSA in the presence and absence of doxycycline (0.1 μg/mL) (bottom). a, e, Bars represent mean ± s.d. a, e, n = 3 biologically independent samples. Statistical significance was determined by two-tailed unpaired t-test.