Extended Data Fig. 1: NHE1 activity regulates pHc and promotes cell cycle progression through early G1.

(a–c) FCS and glucose availability cooperatively regulate pHc. Cells were starved for 24 hours, stimulated with the indicated conditions and pHc was determined. (a) Pooled single cell data of independent experiments N=3, n=30 cells per experiment, two-sided t-Test) are shown as box plot (MATLAB) with median, 25^th and 75^th percentile and whiskers extending to most extreme points indicated. (b) The difference in pHc relative to the starved control (-/-) (b, c) was plotted as mean ± S.E.M. alongside the histogram of single cell measurements. In the histograms, the median pHc was indicated. For all pHc measurements data are pooled single cell data of independent experiments N=3, n=30 cells per experiment, two-sided t-Test. (d) NHE1 is the most abundant NHE localized to the plasma membrane. Relative expression levels of NHE1-9 were determined by qPCR (mean ± S.E.M. of independent experiments, N=3, two-sided t-Test). Subcellular localization of the different NHE proteins is indicated. (e–g) NHE1 activity is required for RB phosphorylation. (e) The fraction of cells with RB phosphorylation at Ser780 from the experiment shown in Fig. 1f is shown (mean ± S.E.M. of independent experiments N= 3, two-sided t-Test). (f) Cells treated as in Fig. 1f were stained for RB^P-Ser807/811 and the fraction of cells with RB phosphorylation (mean ± S.E.M. of independent experiments N=3, two-sided t-Test) (f) and representative images are shown. Scale bar represents 33 µm. (g) Cells were subjected to siRNA against NHE1 and RB phosphorylation was determined after 72 hours by immunofluorescence or western blot. The fraction of RB positive cells (mean ± S.E.M. of independent experiments N=3, two-sided t-Test) and representative images are shown. Scale bar represents 100 µm. (h, i) RPE-1 cells were grown in media of different NaHCO₃ concentration to adjust extracellular pH to the indicated values and relative growth by MTT (mean of independent experiments N=2) (h), the fraction of cells with RB phosphorylation (mean of independent experiments N=2) and representative images (i) are shown. Scale bar represents 100 µm.