Fig. 5: Low extracellular pH reduces the growth inhibitory effect of extracellular ammonia.

A, B A549 cells were cultured for one day in media (DMEM) with 4 mM NH₄Cl and then subjected to indicated treatments for 4 h. DMEM* represents cells cultured in DMEM without NH₄Cl. Western blot analysis of indicated proteins. Bafilomycin A1, 200 nM. LY294002, 10 μM. SAR405, 1 μM. Molecular markers and densitometry analysis are shown in the Source Data file. C A549 cells were cultured for two days in media with the indicated pH and indicated concentration of NH₄Cl. Western blot analysis of indicated proteins. Molecular markers and densitometry analysis are shown in the Source Data file. D A549 cells were cultured for two days in media with the indicated pH and indicated concentration of NH₄Cl. Then, cells were stained with Acridine Orange for 5 min and subsequently analysed by fluorescence microscopy (in vivo). Red squares depict the regions that are magnified to the far right (inserts). E Growth curves based on cell counting. A549 cells were cultured in media with either pH 7.5 or 6.8 and with the indicated concentrations of NH₄Cl. Error bars represent the standard deviation of 3 replicates. *(p < 0.05, two-tailed Student’s t test). F Cell viability assay (CellTiter-Glo). A549 cells were exposed to indicated concentrations of NH₄Cl while being cultured in media with either pH 7.5 or 6.8. Error bars represent the standard deviation of 3 replicates. *(p < 0.05, two-tailed Student’s t test). Cell viability assay. HT29 (G) and A431 (H) cells were cultured in media with either pH 7.5 or 6.8 with the indicated concentrations of NH₄Cl. After three days cell viability was measured by CellTiter-Glo. *(p < 0.05, two-tailed Student’s t test). E–H Source data are provided in the Source Data file.