Figure 2

Pyrazinoylguanidine cytotoxicity is cell type-dependent and most potent for EIPA and HMA. Spheroids of the human cancer cell lines HCT116, BxPC-3, T47D, SKBr-3, MDA-MB-231 and MCF-7 (colon, pancreatic and 4 different breast cancer subtypes, respectively), the non-tumorigenic mammary epithelial cell line, MCF10A and the murine fibroblast cell line, NIH3T3, were grown for 9 days. Spheroids were treated with EIPA (0–10 µM) on day 2, 4, and 7. Spheroid growth was monitored by light microscopic imaging and a viability assay was performed on day 9. (A) Representative images of control- and EIPA- (10 µM) treated spheroids on day 9. Scale bar: 100 µm. Data are from 3 independent experiments. (B) Bar plot displaying cell viability at 10 µM EIPA on day 9 for each cell line. Error bars denote SEM. Data are from 3–9 independent experiments per condition. Dotted grey line represents control spheroids. Statistical significance determined using one-way ANOVA with Dunnett’s multiple comparisons test. **Denote p = 0.0020 for MDA-MB-231 and 0.0017 for MCF10A, while **** denote p < 0.0001 for MCF-7. Values from Fig. 1B,D are included for MCF-7 and MDA-MB-231. (C) Cell viability as a function of EIPA concentration on day 9. Error bars denote SEM. Data are from 3 independent experiments. (D–G) MCF-7 and MDA-MB-231 were grown for 9 days and treated with increasing doses of the respective NHE1 inhibitors amiloride (0–500 µM) (D), DMA (0–80 µM) (E), HMA (0–40 µM) (F) and eniporide (0–20 µM) (G) on day 2, 4 and 7. Graphs display day 9 viability. Error bars denote SEM. Data are from 3 independent experiments.