Fig. 3.

Effects of mTORC1 inhibition on NADPH and G6PD levels in T-ALL cells. (A) Changes in NADPH levels upon 24 h' treatment with 5, 10 or 15 μM everolimus in Jurkat cells (left-hand graph) and T-ALL PDX9, PDX13, and PDX19 cells (right-hand graph). NADPH measurements (pmol NADPH per 10⁶ cells) in treated cells were normalized against values measured in untreated cells. Graphs show mean values and standard error bars from 4 independent experiments, 4 replicates each. (B) G6PD expression in Jurkat (upper panels) and PDX cells (lower panels) treated as described in (A). Shown are immunoblots from representative experiments and plots of the mean G6PD/GAPDH ratios and standard error bars from 6 independent experiments. The right-hand graphs show results of qRT-PCR to measure G6PD mRNA; fold changes in G6PD mRNA in treated versus untreated cells (Eve = 0) were calculated using GAPDH as a housekeeping mRNA. Mean values and standard error bars from 6 experiments are shown. (C) Degradation rates of the G6PD and GAPDH proteins after blocking protein synthesis. Jurkat cells were treated with 10 μM cycloheximide (CHX) alone, with 10 μM cycloheximide + 15 μM everolimus (EVE) or with 10 μM cycloheximide + 15 μM everolimus + 1 μM Vps34-IN1 and harvested for immunoblotting to detect G6PD and GAPDH after 0, 1, 4, 8 and 12 h. The left panel shows immunoblots from a representative experiment. The line graph (middle panel) shows G6PD signals scaled against the value measured in the control (t = 0); resulting values represent the fraction of protein remaining in cells treated with cycloheximide alone (black line), cycloheximide + everolimus (red line) or with cycloheximide + everolimus + Vps34-IN1 (green line). The bar graph (right panel) shows mean values (5 experimental repeats) of G6PD half-life in the different treatments scaled against half-life measured in the cycloheximide alone samples; standard error bars are shown. The indicated pairwise comparisons were statistically significant with the Mann-Whitney test; *** indicates p values < 0.001, ** <0.01 and * < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)