Figure 2.

Metabolic changes triggered by MKK6 expression. 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) Lipid droplet content was quantified 24 h and 48 h after MKK6 induction in the presence or absence of the p38α inhibitor PH797804 (PH). (b) Incorporation of ¹⁴C-glucose into newly synthesized proteins and lipids was analysed 24 h after MKK6 induction, and is represented as fold change towards control. (c) Total lysates from control and MKK6 expressing cells in the presence or absence of PH were analysed by immunoblotting using the indicated antibodies. The uncropped immunoblots are presented in Supplementary Fig. S8. (d) The oxygen consumption rate was analysed 12 h after the induction of MKK6 expression in the presence or absence of PH. Results are presented as pmoles of O₂ consumed per 10⁵ cells. (e) The extracellular acidification rate was measured 12 h after the induction of MKK6 in the presence or absence of PH. Results are presented as pmoles of secreted H⁺ per 10⁵ cells. (f) Lactate production was measured in control and MKK6 expressing cells at 32 h, and is presented as μmol × 10⁶ cells⁻¹ × h⁻¹.