Fig. 3. EGFR-R776H has pH-sensitive proliferation and soft agar transformation.

(A) Immunoblot of lysates from parental NIH3T3 (3T3) cells and cells stably expressing vectors encoding WT or mutant EGFR. (B) pHi in 3T3 cells when cultured with ammonium chloride (NH₄Cl) or EIPA. Data are means ± SEM from four independent experiments. (C) Fold increase in cell number at given pHi for stable WT or mutant EGFR cell lines. Data are means ± SEM from three or more independent experiments, each normalized to parental 3T3 at pHi 7.4. (D) Soft agar transformation assay in NIH3T3 cells stably expressing WT or mutant EGFR or BRAF-V600E. Cells were maintained for 15 days in complete medium or medium supplemented with 5 mM NH₄Cl and then fixed and stained with crystal violet. (E) Colonies >8 pixel² (px²) were counted using ImageJ particle analyzer. Data are from four independent experiments (Tukey box plots). Comparison of 3T3 at pHi 7.2 and 7.4 in (C) used a one-sample t test (two-tailed). All other comparisons in (B), (C), and (E) used Student’s t tests (unpaired, two-tailed) with Holm-Sidak multiple comparisons correction. *P < 0.05, **P < 0.01, ***P < 0.001.