Extended Data Fig. 6: Dissociation between MAPK signalling and differential stimulation of glucose consumption and basal ECAR by KRAS4A versus palmitoylation-deficient KRAS4A and KRAS4B.

a–c, Flp-In T-REx 293 cells were generated that express the indicated KRAS proteins after induction with doxycycline. a, b, Glucose consumption (mean ± s.e.m.; n = 5) (a) and basal ECAR (mean ± s.e.m.; n = 10) (b) were measured in doxycycline-induced cells. This revealed the order of potency to be KRAS4A(G12V/C180S) > KRAS4A(G12V) > KRAS4B(G12V). Significance was determined by Student’s t-test (paired in a; unpaired in b). c, Immunoblot reveals equivalent expression of the three KRAS proteins in the cells used in a and b. Whereas KRAS4A(G12V) and KRAS4B(G12V) induced equivalent levels of phosphorylated (p)ERK and phosphorylated MEK, KRAS4A(G12V/C180S) (which is palmitoylation deficient) was less potent. These cells have constitutively high levels of AKT phosphorylation that were not altered by expression of any form of KRAS. Note also that despite MAPK stimulation, protein levels of HK1 and HK2 were not altered. The immunoblots shown are representative of two independent experiments. t, total. d, Parental HEK293 cells with lower basal levels of phosphorylated AKT were transfected with the indicated constructs, transferred to 0.1% serum 18 h after transfection and lysed 24 h later. Lysates were analysed for the indicated proteins by immunoblot (n = 2).