Annexin 5 fitc propidium iodide staining kit

The annexin V-FITC/propidium iodide (PI) staining kit (BD Biosciences, San Jose, CA, USA) was used to detect apoptosis according to the manufacturer’s protocol. MLE-12 and A549 cells were harvested, resuspended in 150 mL binding buffer, and stained with 5 µL FITC-conjugated Annexin V and 5 µL PI in the dark for 15 min at room temperature. Cells positive for FITC-conjugated Annexin V were detected by flow cytometry (ACEA NovoCyte, San Diego, CA, USA). The data were analyzed using NovoExpress software 1.5. In addition, A549 cells were stained using the TUNEL kit (Roche, Basel, Switzerland), and apoptotic cells were observed using a confocal microscope.

Dimethylsulfoxide (DMSO) was obtained from Sigma-Aldrich (St. Louis, MI, USA). Dulbecco’s modified Eagle medium (DMEM), RPMI 1640 medium, fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco (Grand Island, NY, USA). An Enhanced Cell Counting Kit-8 (CCK-8), a Calcein/PI Live/Dead Viability Assay Kit, Giemsa dye and Reactive Oxygen Species (ROS) Assay Kit were obtained from Beyotime Biotechnology (Shanghai, China). An Annexin V-FITC/Propidium iodide (PI) staining kit and Matrigel matrix were obtained from BD Biosciences (Franklin Lake, NJ, USA). All of the other chemicals used were of analytical grade.

The measurement and quantification of apoptosis in RMC2C cells treated with a drug or combination of drugs was conducted using an Annexin V‐FITC/propidium iodide (PI) staining kit (BD Bioscience). RMC2C cells were first seeded in a six‐well plate and treated with either DMSO (Control), pevonedistat 0.2 μM, carboplatin 20 μM or pevonedistat with carboplatin for 48 h. The cells were then washed in phosphate‐buffered saline (PBS) and harvested by trypsinisation. These harvested cells were washed once with ice‐cold PBS and then with 500 μL of 1× binding buffer. Cells were incubated with FITC Annexin V in 1× binding buffer containing PI in dark at 4°C for 15 min. Following removal of stain, cells were resuspended in 1× binding buffer and then examined using BD FACS Canto II flow cytometry (BD Bioscience, NJ, USA). The obtained flow cytometry data was analysed and graphed using FlowJo_v10.8.1 software.

The three cell lines were plated in 24-well plates (2 x 10⁵ cells/well) and cultured in their corresponding media overnight prior to infection with different MOIs (0.1, 1 and 5) of DENV-2. At 0, 6, 12, 24 and 48 h post-infection, the cells were detached from the wells by incubation in PBS containing 0.1% trypsin and 2.5 mM EDTA (3 min, 37°C), washed once with washing medium, and immediately assessed for viability as follows. Early and late apoptotic cells were determined using Annexin V-FITC/Propidium iodide (PI) staining kit (BD Biosciences) according to the manufacturer's instructions. The apoptotic cells were also analyzed based on caspase-3 and caspase-7 activation using CellEvent Caspase-3/7 Green detection reagent (Thermofisher Scientific Inc.), according to the manufacturer's instructions. The percentages of positively stained cells were determined by flow cytometry and the data were analyzed by Flowjo software.

For biological experiments, Dulbecco’s modified Eagle medium (DMEM), RPMI 1640 Medium, Fetal bovine serum (FBS) and penicillin/streptomycin were bought from Gibco (Grand Island, NY, United States). Dimethylsulfoxide (DMSO) was got from Sigma-Aldrich (St. Louis, Missouri, United States). Enhanced Cell Counting Kit-8, Reactive Oxygen Species (ROS) Assay Kit, Calcein/PI Live/Dead Viability Assay Kit and Giemsa dye were obtained from Beyotime Biotechnology (Shanghai, China). Annexin V-FITC/Propidium iodide (PI) staining kit was bought form BD Biosciences (Franklin Lake, New Jersey, United States).

All the reagents and solvents were obtained from commercially available sources. The ¹H NMR and ¹³C NMR spectra were acquired in a DMSO-d₆ solution using a Bruker 400 MHz or 600 MHz NMR spectrometer.
Dolutegravir-1,2,3-triazole compounds was synthesized in-house. Dimethylsulfoxide (DMSO) was obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Dulbecco’s modified Eagle medium (DMEM), RPMI 1640 Medium, Fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Gibco (Grand Island, NY, USA). Enhanced Cell Counting Kit-8, Calcein/PI Live/Dead Viability Assay Kit, Giemsa dye and Reactive Oxygen Species (ROS) Assay Kit were obtained from Beyotime Biotechnology (Shanghai, China). Annexin V-FITC/Propidium iodide (PI) staining kit and Matrigel Matrix were provided by BD Biosciences (Franklin Lake, New Jersey, USA).
Human lung cancer cell lines A549, PC-9 and H460 were all obtained from ATCC. Cells were cultured in DMEM or RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin at 37 °C with a 5% CO₂-humidified atmosphere.