Extended Data Fig. 7: pHc is regulated by Akt activity and active glucose metabolism.
From: Cytosolic pH regulates proliferation and tumour growth by promoting expression of cyclin D1
(a) Cells as in Fig. 3g were grown and relative biomass accumulation was scored by MTT 72 hours after DMA treatment. (mean ± S.E.M. of independent experiments N=6, two-sided t-Test). (b) Cells as in Fig. 3g were grown and RB phosphorylation was scored 24 hours after DMA treatment. (mean ± S.E.M. of independent experiments N=3, two-sided t-Test). (c) CREB1 peptides interact with the KIX domain of CBP. A result of a pull-down of the KIX domain of CBP with biotinylated peptides representative for 6 independent experiments is shown. (d) RPE-1 cells were starved, stimulated with the indicated conditions and changes in cytosolic pH (mean ± S.E.M. of pooled single cell data from independent experiments, N=3, was plotted alongside the histogram of single cell measurements, n=30 cells per experiment) was determined. (e) RPE-1 cells were starved, stimulated with FCS and glucose in the presence of DMA and C646 and Akt-dependent phosphorylation of TSC2 was determined by western blotting. Blots representative of three independent experiments are shown. (f) Elevated pHc requires active glucose metabolism. pHc as determined in Fig. 3j is plotted from pooled single cell data of independent experiments N=3, n=30 cells per experiment are shown as box plot (MATLAB) with median, 25th and 75th percentile and whiskers extending to most extreme points not considered outliers, and outliers (+) indicated, two-sided t-Test.
