Figure 1.

MKK6 expression makes cells highly dependent on glucose for survival. U2OS cells expressing a Tet-regulated construct were either mock treated (control) or treated with tetracycline for the indicated times to induce the expression of constitutively active MKK6. (a) Total cell lysates were analysed by immunoblotting using the indicated antibodies. Phosphorylated p38α and p38γ are indicated by arrowheads. The uncropped immunoblots are presented in Supplementary Fig. S8. (b) Control and MKK6 expressing cells were incubated in complete media or glucose free media for 24 h and cell survival was assayed using Annexin V/PI staining. Live cells were determined as the cell population Annexin V⁻ and PI⁻. (c) Morphology of cells cultured in complete or glucose free media for 24 h. Bars = 50 μm. (d) Glucose consumption by control, MKK6 expressing cells and MKK6 expressing cells treated with the p38α inhibitor PH797804 (PH) were measured at the indicated times and presented as μmol × 10⁶ cells⁻¹ × h⁻¹. (e) Control and MKK6 expressing cells were grown in the presence or absence of the p38α inhibitor PH797804 (PH) for the indicated times and the expression levels of mRNAs encoding glycolytic genes and GLS1 was analysed. Results are presented as fold change towards control. (f) Glutamine consumption was determined as in (d). (g) Control and MKK6 expressing cells were grown for 24 h in complete (+Glc) and glucose depleted media (−Glc) supplemented with 2 mM pyruvate or 1 mM dimethyl-2-ketoglutarate (αKG), as indicated, and cell survival was assayed using Annexin V/PI staining.