Fig. 1.

Effect of SAM, 25(OH)D, and their combination in vitro. a Schematic of the in vitro treatment protocol. b Human (ZR-75-1, MDA-MB-231) and murine (PyMT-R221A, E0771) breast cancer cells were treated with vehicle alone control, SAM (200 µmol·L⁻¹) or 25(OH)D (100 nmol·L⁻¹) alone and SAM + 25(OH)D every other day; and cell number was determined using Coulter counter on days 1,3, and 5 post-treatment. Results are shown as the mean ± SEM from at least five independent experiments. Significant differences from the control groups in each cell lines were determined using ANOVA followed by post hoc Tukey’s test and are represented by asterisks. c Following treatment, 5 × 10³ cells from the control and different treatment groups were subjected to clonogenic survival assay. The culture media was refreshed every 3–4 days for a period of about 2 weeks, the cells were then stained with crystal violet, and the total number of colonies was counted under the microscope. Results are shown as the mean ± SEM of at least five independent experiments. Significant differences were determined using ANOVA followed by post hoc Tukey’s test and are represented by asterisks