Cellquest software version 5

HLE-B3 cell apoptosis was analyzed using an Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (BD Biosciences). Briefly, 1x10⁴ HLE-B3 cells/well were cultured in six-well plates, digested using 0.25% trypsin without EDTA and resuspended in 500 µl Annexin binding buffer. Subsequently, the cells were incubated with 5 µl Annexin V-FITC and 5 µl PI in the dark for 15 min. Apoptotic cells were analyzed using a fluorescence-activated cell sorting system (FACSVantage; BD Biosciences) and CellQuest software (version 5.1; BD Biosciences).

Min6 cell apoptosis was measured by performing flow cytometry. Min6 cells (10⁶) were digested using 0.2% trypsin, followed by washing with pre-cooled PBS. Cells were stained using an Annexin V-FITC/PI kit (cat. no. 70-AP101-100; Multisciences (Lianke) Biotech Co., Ltd.) according to the manufacturer's protocol. In brief, Min6 cells were stained with 5 µl Annexin V-FITC and 10 µl PI at room temperature for 5 min in the dark. Apoptotic cells (early and late apoptosis) were analyzed using a FACSCalibur flow cytometer (BD Biosciences) and CellQuest software version 5.1 (BD Biosciences).

The apoptosis rate was detected by Annexin V-FITC/PI apoptosis kit (eBioscience; Thermo Fisher Scientific, Inc.). SKOV3 and OVCAR3 cells after transfection for 48 h were seeded into 6 well plates at 2×10⁵ cells/well. After the cell confluence reached 80%, the cells were collected. Then, 400 µl premixed 1X buffer binding was added into the cells, and the cell suspension was transferred into a flow tube. Next, into the cell suspension in the flow tube was added 5 µl of Annexin V-FITC and 5 µl of propidium iodide (PI) to incubate for 15 min in the dark. After incubation, the cells were analyzed using the BD FACScalibur Flow cytometry (BD Biosciences), which is equipped with Cell Quest software version 5.1 (BD Biosciences).

Following transfection of the miRNA inhibitor into the hypoxic environment and incubation at 37°C for 48 h, JAR cells were collected by trypsin digestion method and washed with PBS prior to re-suspension in 250 µl DMEM. Cold (4°C) dehydrated ethanol (99%) was added to this buffer and incubated overnight at 4°C. Following treatment, cells were collected and incubated with 200 µl PI (20 µg/ml) using a cell cycle assay kit (Vazyme Biotech, Co., Ltd., Nanjing, China) at 37°C for 15 min. Samples were immediately analyzed using flow cytometry (EPICS^® XL™; Beckman Coulter, Inc.). Data acquisition and analysis were performed using CellQuest software version 5.1 (BD Biosciences) (23 (link),24 (link)).

ROS were detected in different groups (Control, H₂O₂, LBP1 + H₂O₂, LBP2 + H₂O₂, LBP3 + H₂O₂) with a 2′,7′-dichlorofluorescin diacetate (DCFH-DA) assay (Beyotime Institute of Biotechnology). After 6 h of 250 µmol/l H₂O₂ treatment, cells, treated with different concentrations (100, 200 and 400 µg/ml) of LBP for 6 h, were seeded into wells of 6-well plate. DCFH-DA (10 µmol/l) was subsequently added into each well. After incubation for 20 min at 37°C, cells were rinsed with PBS and analyzed by flow cytometry. ROS levels were analyzed by CellQuest software version 5.1 (BD Biosciences, Franklin Lakes, NJ, USA) and results were calculated relative to the control group.