Modfit software

Collect each group of test cells, wash the cells with pre-cooled PBS, add 300 μl of binding buffer to resuspend the cells, and adjust the cell density to 1 × 10⁶ cells/ml. According to the manufacturer’s recommendations, Annexin V-FITC/PI Apoptosis Detection Kit (TransGen Biotech) was used for apoptosis analysis. Take 100ul of cell suspension into the flow tube, and add 5ul each of Annexin V-FITC and PI. After mixing, incubate at room temperature for 15 min in the dark, and add 400 ul of PBS to the reaction tube. Ensure that the cell apoptosis detected by flow cytometry within 1 h. The ModFit software (Verity Software House) used to analyze flow cytometry data.

The cells were resuspended at a density of 2 × 10⁶ cells/mL in buffer containing 1× PBS, 2 mM EDTA and 0.5% bovine serum albumin (BSA), and incubated at 4 °C in dark for 10 min with fluorescein isothiocyanate (FITC) conjugated CD326 antibody (MACS, Miltenyi Biotec). After centrifugation and washing, cells were resuspended in 200 μl buffer and analyzed for the expression of EpCAM with FACSCalibur flow cytometer (Becton–Dickinson, Franklin Lakes, NJ), and data were analyzed using ModFit software (Verity Software House, Topsham, ME). At least 20,000 cells were analyzed per sample.

Rat glomerular MCs were seeded in 24-well plates at a density of 5,000 per well in medium containing 10% FBS with 5.5 mM D-glucose. Six replicate wells were used for each sample at each time point. At the indicated time point, medium was removed and serum-free medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 0.5 mg/ml) was added into each well. Four hours after incubation at 37°C, cellular formazan product was dissolved with acidic isopropanol, and the absorbance at 570 nm was measured by spectrophotometry (Beckman Du640B). For the cell cycle flow cytometry assay, cells were digested by trypsin-EDTA, harvested as many as 1×10⁶ cells, and fixed in 70% ethanol at 4°C. After 12 h, cells were centrifuged (1,000×g, 5 min, 4°C), resuspended in PBS containing 0.05 mg/ml RNase A (Sigma), then incubated at room temperature for 30 min. After washing and staining with 10 mg/ml propidium iodide, we filtered the cells using a 60-μm mesh. At the last, 10,000 cells were analyzed by flow cytometry (FACSCalibur, BD Company) with ModFit software (Verity Software House, Inc.).

MDA-MB-231 cells were seeded (2 × 10⁵ cells/ml) into 6-well plates and treated with either 10μM ICG-001 or 2μM JA for 48 h. After treatment, cells were washed with PBS, fixed in chilled 75% ethanol, treated with RNase and then stained with PI solution (50 μg/ml). Cell cycle distribution was analyzed with the FACSCalibur analyzer (BD Biosciences, Franklin Lakes, NJ) and Cell-Quest software. The percentage of DNA content at different phases of the cell cycle was analyzed with Modfit-software (Verity Software House, ME, USA).