Fig. 1: Extracellular ammonia reduces the growth of cancer cells in a dose-dependent manner.

A Growth curves for indicated cell lines cultured in media with indicated concentrations of NH₄Cl. Error bars display the standard deviation of biological triplicates. *(p < 0.01, two-tailed Student’s t test). Source data are provided in the Source Data file. B–E Indicated cell lines were cultured in media with indicated concentrations of NH₄Cl for three days. Then, cells were subjected to 10 min of BrdU (20 μM) pulse labelling. Cells were subsequently analysed for BrdU and DNA content (PI, propidium iodide) by flow cytometry. The experiment was performed three times, yielding similar results. B Dot plot showing DNA content (PI), and BrdU incorporation (BrdU) for HT-29 cells. C, D Column chart showing the distribution of cells in each subpopulation defined in (B). E Histograms depicting BrdU incorporation in cells replicating DNA (S-phase cells). Grey histograms represents cells that have been cultured without NH₄Cl. F A549 cells were grown in the presence of the depicted concentrations of NH₄Cl for three days. Then, cells positive for Histone H3 phosphorylated at ser10 (p-H3(ser10)) were registered by immunofluorescence. Where indicated, cells were treated with 100 ng/ml Nocodazole to arrest cells in M-phase. Error bars represent the standard error of the mean. *(p < 0.05, as determined by two-tailed Student’s t test). Source data are provided in the Source Data file.