Modfit software

Transfected cells were collected in an exponential growth phase and were ethanol fixed. 1×10⁴ transfected MKN-45cells, including negative control ones, were respectively cultured in 96-well microtiter plates in triplicate and incubated for 5 days at 37°C with an atmosphere of 5% CO₂. OD was measured by WST (Water-soluble tetrazolium salt USA) assay by using CCK8 Assay Kit (Dojindo, Japan) according to the protocol. The curves of cell proliferation were plotted.
RNaseA treatment and Propidium Iodide staining were carried out. Cells were detected under flow cytometry by FACSCalibur (Becton Dickinson, USA). Cell populations at the G0/G1, S and G2/M phases were quantified by Modfit software (Becton Dickinson, USA) excluding a calculation of cell debris and fixation artifacts.

After 24 h of treatment with compound 11 at 50 µM (6-well plates, 5 × 10⁴ cells/mL), LNCaP cells were collected and washed with PBS and resuspended in 450 µL of a cold solution of 0.5% bovine serum albumin (BSA; Amresco, Solon, OH, USA) and 1 mM EDTA in PBS, followed by fixation with 70% EtOH and kept at −20 °C. Afterward, fixed cells were washed twice with PBS and resuspended in a solution of PI (50 µg/mL) prepared in 0.5% BSA and 1 mM EDTA in PBS and then incubated with Ribonuclease A from bovine pancreas at a final concentration of 0.5 µg/µL (solution in 50% glycerol, 10 mM Tris-HCl, pH 8; Sigma Aldrich, St Louis, MO, USA) for 15 min in the dark. For comparison, untreated cells were used as negative control and cells treated with 5-FU at 50 µM were used as positive control. Each experiment was performed in duplicate and independently repeated. A minimum of 10,000 events was acquired using BD Accuri Software and analysis was performed by Modfit software (Becton Dickinson, San Jose, CA, USA).

The proliferation abilities of 5-aza-treated SGC-7901, MKN-45 and their NC cells were determined by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylte-trazolium bromide) assay using a Cell Proliferation Kit 1 (Roche Applied Science), according to the manufacturer’s instructions. For cell cycle analysis, cells were treated by ethanol fixation, RNase A, and propidium iodide (PI) staining, and then detected by flow cytometry using a FACSCalibur (Becton–Dickinson, USA). Cell populations in the G0/G1, S, and G2/M phases were quantified by Modfit software (Becton–Dickinson), allowing for cell debris and fixation artifacts.

The cell cycle analysis was employed by propidium iodide staining and quantified by a FACS Calibur flow cytometer (Becton, Dickinson, San Jose, CA, USA)[46 (link)]. The percentage of cells in each phase of the cell cycle was analyzed with Modfit software (Becton, Dickinson, San Jose, CA, USA).

Cell cycle analysis was performed on sub-confluent cell cultures using CycleTest™ Plus DNA Reagent Kit (Becton Dickinson). The test was carried out with synchronous cells. DNA was stained with PI according to the manufacturer’s instructions. Tumor cells were then subjected to flow cytometry using FACScan. For each sample, 10,000 events were observed. Data acquisition was carried out using the CellQuest software, and the ModFit software (Becton-Dickinson) was used to calculate the cell cycle distribution. The number of gated cells in the G1, G2/M, or S phases was expressed in percentage form.

Naive or activated splenocytes were incubated with different treatments for 48 h, as in a previous study [15 (link)], the 1 × 10⁶ cell pellets were washed with PBS and fixed in 3 ml of 70% ethanol at 4 °C for 30 min. After centrifugation at 400 × g, the fixed cells were resuspended in 1 ml of DNA staining buffer (containing 5% Triton-X 100, 0.1 mg/ml RNase A and 4 μg/ml propidium iodide) and incubated for 30 min at room temperature. Ten thousand cells were analyzed for DNA content using a Becton–Dickinson FACScan (Becton Dickinson, Mountain View, CA), and the cell cycle distribution was determined using ModFit software (Becton Dickinson).