Extended Data Fig. 8: NHE1 activity is critical for cell-cycle progression in PDAC cell lines.
From: Cytosolic pH regulates proliferation and tumour growth by promoting expression of cyclin D1
(a) Cells of the indicated genotype expressing pHluorin were starved for 24 hours and pHc was determined 30 min following stimulation with the indicated conditions. For all pHc measurements data are pooled single cell data of independent experiments N=3 for PANC-1, N=3 for MIA Paca-2), n=30 cells per experiment, two-sided t-Test. Box plot (MATLAB) with median, 25th and 75th percentile, whiskers extending to most extreme points not considered outliers, and outliers (+) indicated. (b) Cells as in (a) were starved, treated with DMA for 6 hours and cyclin D1 and RB phosphorylation were determined by western blotting. (c) Cells were subjected to siRNA against NHE1 and cyclin D1, RB phosphorylation and NHE1 abundance were determined by western blotting. (d) Cyclin D1, but not NHE1 expression correlates with survival in PDAC cell lines. Data obtained from TCGA were independently analyzed for a correlation of cyclin D1 and NHE1 mRNA and survival and Kaplan Meier analysis was performed using log-rank test (N=85 patients with high expression of NHE1 or cyclin D1, respectively, N=96 patient with low expression of these markers). Hazard Ratio for NHE1 (with confidence interval) HR = 1.08 (0.72 - 1.62); for cyclin D1 HR = 2.05 (1.36 - 3.10).
