Giemsa stain

For the colony formation assay or clonogenic assay, cells were seeded at 10⁶ in two 6 cm dishes per cell line per dose (0, 2, 4, 6, 8, 10 Gy). The following day, the plates were irradiated at the appropriate doses. The cells were collected and resuspended in media and then counted to be seeded into 6 cm dishes for each dose and cell line to achieve approximately 50 visible colonies at the end of the experiment. The cells were then incubated for 2 weeks. Then, cells were washed with Phosphate Buffered Saline (PBS, Sigma-Aldrich, UK) and fixed using 2 mL of 10% neutral buffered formalin (Fisher, UK). The plates were then washed in PBS and allowed to air dry before the Giemsa staining solution was added and incubated for 5 hours at room temperature. Before imaging the plates were washed with water and counting of colonies was carried out. The Giemsa staining solution was made by mixing 6.4 mL of 67 mM sodium phosphate buffer, pH 7.0, 5 mL of Giemsa stain (Abcam, UK) and water. The phosphate buffer, pH 7.0 was made using 1 : 2 ratio of 1 M monobasic sodium phosphate solution and 1 M dibasic sodium phosphate solution.

HCCLM3 or MHCC97H cells (2 × 10³) were cultured into 6-well plates. After 24 hrs, cells were cultured under tripterine (0.3 μM, 1 μM or 3 μM). After 14 days, the cells were fixed with 4% Polyoxymethylene and stained using Giemsa Stain (Abcam, Cambridge, UK). Afterwards, the cell colonies were counted directly with the naked eyes.