Pi flow cytometry kit

For cell cycle analysis, maltol- or cisplatin-treated B16F10 cells were fixed in ice-cold 70% ethanol overnight at 4°C. Then, the cells were incubated in PBS buffer containing RNase (100 μg/mL) and propidium iodide (PI, 50 μg/mL) for 30 min at room temperature, utilizing a PI flow cytometry kit (abcam, Cambridge, United Kingdom). For apoptosis analysis, B16F10 cells treated with maltol or cisplatin were stained with annexin V and PI using a FITC annexin V apoptosis detection kit with PI (Biolegend, San Diego, CA, United States). Cell surface PD-L1 detection was performed with reference to previous protocols (Tang et al., 2018 (link); Xu et al., 2018 (link)). Maltol or cisplatin-treated B16F10 cells were first blocked with anti-CD16/32 (anti-FcγIII/II receptor, clone 2.4G2, BD Biosciences, ≤ 1 µg/million cells), then incubated with PE-conjugated anti-mouse PD-L1 antibody (12-5982-83, eBioscience, San Diego, CA, United States, 1:200) in FACS buffer (PBS containing 0.1% BSA and 0.02% NaN₃) for 30 min at 4°C. Following PBS washes, the samples were resuspended in FACS buffer and analyzed through flow cytometry.

Cell cycle phases of HepG2 cells were analyzed using Propidium Iodide (PI) Flow Cytometry Kit (ab139418, Abcam, Cambridge, UK). In six-well plates, cells were seeded at a density of 1 × 10⁵ cells per well and incubated for 24 h. The cells were then treated to the predetermined IC₅₀ values for 48 h. After that, the cells were detached and collected via centrifugation at 800 × g for 5 min, before being washed and resuspended with 1 mL of PBS. After a second centrifugation at 800 × g for 5 min, the cells were fixed with ethanol (70%) and stored at 4 °C for 2 h. After washing with PBS, the pellet was treated with RNase and further stained with 200 µL of PI at 37 °C in dark for 30 min. The cellular DNA content was determined at 488 nm using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Acquired data were examined utilizing the CellQuest Software (BD Bioscience, San Jose, CA, USA).

To test the effect of SR1078 on cell viability, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out as previously described^{61 (link)}. Cell proliferation was assessed using the CCK-8 Kit (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, 3–5 × 10³ cells/well were seeded in 96-well plates. At determined times, CCK-8 reagent (10 µl) was added to the wells and incubated at 37 °C for 4 h, and absorbance (450 nM) was measured. Caspase 3/7-mediated apoptosis was determined using the Caspase-Glo assay kit (Promega). Briefly, 5 × 10³ cells/well were seeded in 96-well plates and subjected to different treatments for 24–48 h. Equal volumes of caspase reagent were added to the wells, and luminescence was measured in a plate-reading luminometer after 3 h incubation. Cell cycle distribution was measured by PI Flow Cytometry Kit (abcam# 139418). LAN5 RORα cells were harvested and fixed (70% ethanol for 2 h). Cells were stained with 50 µg/ml PI for 30 min before FACS analysis (BD LSRII with BD FACSDIVA v6.1.3). Cell population (%) in G0-G1, S, and G2 phases was analyzed by FlowJo v7.6.1.

Cell cycle assessment was performed 24 h post injury to achieve a snapshot of the cell state after their respective treatments. Analysis was performed utilizing the propidium iodide (PI) flow cytometry kit (Abcam, Cambridge, MA, USA), essentially following the manufacturer’s protocol. Briefly, cells were lifted with TrypLE Express (Gibco, Grand Island, NY, USA) treatment for 5–10 min at 37 °C. The duration of the cell lifting varied in the experiment but was monitored by eye periodically for cell sheet lifting. Lifted cells were pelleted with 500 g for 5 min and subsequently fixed by resuspension in 400 µL of ice-cold PBS followed by addition of 800 µL of ice-cold 100% EtOH. On the day of analysis, the fixed cells were pelleted at 500 g for 5 min followed by resuspension in 300 µL of a 1× PI solution with 1× RNase at 37 °C for 30 min. The cells labeled with PI were then analyzed using a flow cytometer (BD FACS Calibur, Franklin Lakes, NJ, USA) with a 488 nm excitation and the FL-2 emission filter. Cell-cycle gates were selected manually on one representative trace for the experiment and then the same gates were applied across samples to calculate the percentages of cells in certain stages of the cell cycle. Figure S4 shows the gating strategy, which involved gating fixed cells by size and granularity, excluding debris, and defining the cell cycle phases for G1, S, and G2-M.

Apoptotic cells were identified using the Propidium Iodide (PI) Flow Cytometry Kit (abcam) and FITC Annexin V Apoptosis Detection Kit I (BD) according to the manufacturer's instructions. NKTCL cells were transfected for 24 hours and then cultured for 72 hours in a humidified atmosphere (37°C, 5% CO₂) before determining the cell cycle and the extent of apoptosis. The cells were collected, washed twice with cold PBS and resuspended in 1× binding buffer. For cell cycle analysis, the cells were stained with 5 μL PI for 10 minutes in the dark at room temperature. For the cell apoptosis analysis, the cells were stained with 5 μL annexin v‐FITC for 15 minutes and then 5 μL PI for 10 minutes in the dark at room temperature. The cells were examined using a FACSCanto II flow cytometer (BD Biosciences).