Coomassie brilliant blue solution

MUC-1 cells (2 × 10³ cells/well) were seeded in 12-well plates and allowed to grow in the absence or presence of XCT790 (1, 5, 10 µM) for 14 days. Colonies were stained and fixed with Coomassie Brilliant Blue Solution containing methanol (Sigma) for 10 min. Colonies (>50 cells) were counted by Image J (NIH) software.

The cells were collected one hour after irradiation using the Accutase solution, resuspended in a culture medium, and counted with a MOXI Z automated cell counter (Orflo Technologies, Ketchum, ID, USA). Cells were plated onto T-25 flasks with vented caps at 500 cells for all of the cell locations (controls plus intestine, lung, thyroid, and brain), except for the prostate, for which 4000 cells were used. Cells from each biological replicate were plated in three replicates and cultured in RPMI 1640 with 10% FBS for 16 days. Next, cells were fixed in ethanol and stained using Coomassie Brilliant Blue solution (Sigma-Aldrich, St. Louis, MO, USA). To determine the colony number, the flasks were photographed using the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA) and analysed with Image Lab Software v6.0 (Bio-Rad, Hercules, CA, USA). Colonies were scored by counting clusters with ≥50 cells. Plating efficiency (PE) was determined by calculating the ratio between the number of colonies and number of plated cells. The surviving fraction was the ratio between the PE values of the irradiated and control cells.

H295R, SW-13 and MUC-1 cells were treated with increasing doses (0, 10, 20 μM) of curcumin for 24 h. Cells were grown in 6-well plates until about 80–90% confluency was reached and then a 10μL pipette tip was used to create a scratch/wound with clear edges across the width of a well. Cells were then stained with Coomassie Brilliant Blue solution (Sigma) for 10 min and photographs were acquired under an inverted phase contrast microscope (Olympus CKX53) with 10X objective at 0 h and 24 h.