Figure 6.

MK2 mediates the MKK6-induced metabolic changes. 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 and b) Glucose consumption (a) and glutamine consumption (b) by control, MKK6 expressing cells and MKK6 expressing cells treated with the MK2 inhibitor PF3644022 (MK2i) were measured at the indicated times and presented as μmol × 10⁶ cells⁻¹ × h⁻¹. These samples were processed in parallel and using the same control and MKK6 expressing cells as the samples presented in Fig. 1d and f. (c) The expression levels of mRNAs encoding proteins involved in glycolysis and glutaminolysis were determined by qRT-PCR after MKK6 induction in the presence or absence of the MK2 inhibitor III (MK2i-III) for the indicated times. (d) Total cell lysates were prepared from control and MKK6 expressing cells in the presence or absence of the p38α inhibitor PH797804 (PH) or the MK2 inhibitor PF3644022 (MK2i), and were analysed by immunoblotting using the indicated antibodies. The uncropped immunoblots are presented in Supplementary Fig. S8. (e) Control and MKK6 expressing cells were grown in glucose-deprived conditions in the presence or absence of MK2i 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⁻.