Cell cycle assay kit

For apoptosis assay, cells were stained with an Apoptosis Assay Kit (Wanlei Bio, Shenyang, China) according to the manufacturer’s instructions, and then detected by fluorescence-activated cell sorting (FACS) analysis. Briefly, cells were seeded in 6-well plates for lentivirus infection. Forty-eight hours later, cells were harvested and washed twice with cold phosphate buffered saline (PBS; double-helix, Shanghai, China). Subsequently, cells were incubated with 5 μl Annexin V-Light 650 and 10 μl propidium iodide (PI) in the dark for 15 min at room temperature. The apoptotic cells were detected by FACS analysis (Accuri C6, BD Biosciences) and the data were analyzed using BD Accuri C6 Software (BD Biosciences) on 10,000 events.
For cell cycle assay, cells were labeled with PI using a Cell Cycle Assay Kit (Beyotime) according to the manufacturer’s procedures. Briefly, 48 h after lentivirus infection, cells were harvested, washed twice with cold PBS, and fixed with ice-cold 70% ethanol for 2 h. Fixed cells were subsequently treated with 25 μl PI. Finally, 10 μl ribonuclease (RNase A) was added to the cells. The DNA content was then quantitated by FACS Accuri C6 (BD Biosciences), with an excitation wavelength of 488 nm and an emission wavelength of 625 nm. Data were analyzed using BD Accuri C6 Software (BD Biosciences) on 10,000 events.

The cells were divided into six groups: CON (no palmitate), PA, PA + SIT-L, PA + SIT-H, PA + PBA, or TM for 24 h, respectively, then cells were harvested by trypsin, centrifuged at 1,000×g for 5 min, resuspended in ice-cold PBS, and fixed in 70% ethanol at 4°C for 18 h. The fixed cells were washed again using ice-cold PBS and incubated with 500 μl propidium iodide containing 0.05% RNase A for 30 min at room temperature in the dark. Finally, a cell cycle distribution profile was detected on BD FACS Verse flow cytometer (United States) at the excitation wavelength of 488 nm. The cell cycle assay kit was from Beyotime Institute of Biotechnology (China). The percentages of cells in G₀/G₁, S, and G₂/M phases were analyzed with Flowjo 7.6 software. The experiment was repeated three times.

Rhein (batch No. 5045, purity >98.0%) was purchased from Shanghai Standard Biotech Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS), 0.25% trypsin, penicillin, and streptomycin solution were obtained from Corning (Corning, NY, USA). RPMI 1640 medium, dimethyl sulfoxide (DMSO), PBS, and MTT were purchased from Solarbio (Beijing, China). The LDH Assay Kit, Annexin V-FITC Apoptosis Assay Kit, DAPI Assay Kit, MMP Assay Kit, ROS Assay Kit, and Cell Cycle Assay Kit were obtained from Beyotime (Nanjing, China). Reduced glutathione (GSH) was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Antibodies against Fas (#4233S), Bax (#5023T), Bcl-2 (#15071), p53 (#2524T), p21 (#2947T), cyclin A (#4656T), CDK 2 (#2546T), cleaved caspase-3 (#9661T), cleaved caspase-9 (#9501T), cytochrome c (#4280T), and PARP (#9542T) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody against caspase-8 (#ab25901) was purchased from Abcam (Shanghai, China) (#ab25901).

A flow cytometry approach was applied to determine cell apoptosis and cell cycle. An annexin V-FITC/propidium iodide cell apoptosis detection kit (BD Biosciences, USA) was used to detect cell apoptosis. U251 and U87 cells were trypsinized and re-suspended in 500 μL binding buffer, then 1 μL annexin V-FITC and 1 μL propidium iodide were added to the cells for 25 minutes of incubation. Cells were washed once with the binding buffer and then analyzed on the Accuri Cytometer (BD Biosciences). The cell cycle assay kit (Beyotime, China) was used to assess the cell cycle. U251 and U87 cells were fixed with 75% anhydrous ethanol overnight in the fridge. After RNase digestion, the samples were stained using propidium iodide for 30 minutes in the dark and then analyzed on the Accuri Cytometer (BD Biosciences).

The cells were fixed in 70% ethanol at 4 °C overnight and then stained with Cell Cycle Assay Kit (C1052, Beyotime, China) for 10 min. All the cells were analyzed by flow cytometry (BD FACSCalibur, Franklin Lakes, NJ, USA).

A cell cycle assay kit (Beyotime, Shanghai, China, C1052) was used to detect cell cycle changes in different generations of ESCs. The collected ESCs were re-selected with 70% ethanol and fixed at 4 °C for 30 min. After, the cells were resuspended with 70% ethanol and fixed at 4 °C for 30 min. After the cells were collected using centrifugation, 500 μL PI staining solution was added, and it was incubated at 37 °C for 30 min without light. The stained cells were detected using flow cytometry.