Wst 8 assay

Cell proliferation was analyzed following a previously described protocol (13 (link)). Briefly, A172 cells were seeded at a density of 5×10³ cells/well onto 6-well plates and cultured in DMEM with 10% FBS at 37°C for 24 h. Cells were then treated with SB (0, 0.5, 1, 2 and 4 mM) for 7 days. Following 4 days treatment, a subset of A172 cells treated with 2 mM SB were washed with PBS and cultured for a further 4 days in DMEM with 10% FBS without SB. For all cells, the culture medium was replenished every 2 d and cell numbers were recorded every 24 h using a WST-8 assay (cat no. 341-07761; Wako Pure Chemical Industries, Ltd.). Cell cycle analysis was performed using a BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's protocol. Briefly, A172 cells were cultured with indicated concentrations of SB, 10, 100 nM TSA or 0.1, 1 mM methotrexate for 4 d, then collected and resuspended in 300 µl of PBS. After, 700 µl of 100% ethanol was added and kept on ice for 30 min to fix the cells. The fixed cells were resuspended in PBS supplemented with 100 µg/ml of RNase A and 50 µg/ml of propidium iodide (PI) at room temperature for 1 h. The PI stained cells were analyzed according to standard FACS protocol for cell cycle analysis with BD FACStation (v6.1, BD Biosciences, Franklin Lakes, NJ, USA).