Cell culture and chemicals
The OSCC cell lines (HSC3, HSC4) were obtained from Hokkaido Univ. (Hokkaido, Japan) and cultured in DMEM/F12 medium (WELGENE, Gyeongsan, Republic of Korea) with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin) at 37°C in an incubator with 5% CO2. All experiments were done with cells cultured at 50–60% confluence. PPT was purchased from Sigma-Aldrich (P4405, St Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO) and stored at − 20°C.
Trypan Blue Exclusion Assay
The assay was used to determine the effect of PPT on cell viability. The OSCC cells were seeded into 6-well plates and incubated with 100 nM of PPT for 24 h, followed by staining with 0.4% trypan blue (Gibco, Paisley, UK). The viable cells were counted using a Corning® Cell counter. All experiments were performed independently 3X with triplicate samples for each experiment.
Assay For Cell Proliferation
To assess cell proliferation, the cells were seeded into 96-well plates and treated with 100 nM of PPT for 24 h. Then, 10 µl of Cell Counting Kit-8 solution (#CK04-500; Dojindo, Japan) was added to each well, thoroughly mixed with the culture media, and incubated 2 h at 37°C. The optical density (OD) of each well's depth was assessed using a microplate reader (Hidex, Turku, Finland) at 450 nm and the OD values are reported as the mean ± SD.
Soft Agar Assay
Basal Medium Eagle (BME) with sodium carbonate was prepared by dissolving in sterile distilled water a 2X BME solution, which was then purified using a 0.2 µM syringe filter. The bottom agar (0.5%) was prepared by mixing 2X BME, L-glutamine, gentamicin, phosphate-buffered saline (PBS), FBS, and 1.25% agar. The wells were precoated at 3 ml per well along with 0.3% top agar. After curing at room temperature (RT) for 1 h, 200 µl of culture medium was added every 3 days with DMSO and 100 nM PPT and the cells were incubated at 37°C for 10 to 14 days. Images of the colonies were randomly captured (4 photos per well) under a microscope (Olympus, Tokyo, Japan) at a 40× magnification. The number of colonies with varying diameters was determined using Image J software.
Analysis Using Flow Cytometry (Cell Cycle Analysis)
After treatment with PPT, HSC3 and HSC4 cells were collected, resuspended, washed with PBS, and fixed in 70% ethanol overnight at − 20°C. Incubation of the cells with a 20 g/ml propidium iodide solution (P4170, Sigma-Aldrich) and RNase A (20 µg/ml) for 15 min at 37°C. Cell cycle distribution was determined using an LSRFortessa™ Cell Analyzer (BD Biosciences). A minimum of 10,000 cells were analyzed for each sample using BD FACSDIVA™ software. The percentage of the sub‑G1 fraction was by using FlowJo software version 9/10 to quantify.
Annexin V/pi Double Staining
Apoptosis was assessed using an FITC‑Annexin V Apoptosis Detection Kit (BD Pharmingen) based on the manufacturer's instructions. The floating and attached cells were gathered, washed with ice-cold PBS twice, and spun down in a centrifuge (180 × g, 5 min, 4°C). The cells were then resuspended in Annexin V binding buffer and treated with 3 µl of Annexin V-FITC at RT and in the dark for 15 min followed by the addition of 1 µl of PI. The cells were then transferred to a FACS tube and analyzed by flow cytometry using an LSRFortessa™ Cell Analyzer (BD Biosciences). The Annexin V(+)/PI(−) staining in cells meant early-stage apoptosis, while Annexin V(+)/PI(+) staining meant late-stage apoptosis. The percentage of cells displaying Annexin V/PI staining was determined by analyzing a minimum of 10,000 cells using BD FACSDIVA™ software.
Mitochondrial Membrane Potential (ΔΨm) Assay
ΔΨm was measured via employing the lipophilic fluorescent dye JC-1 in flow cytometry and the MitoScreen kit (BD Pharmingen). The cells were trypsinized, then flushed with PBS and collected by centrifugation at 3500 rpm for 5 min. The cell pellets were resuspended in 1X JC-1 working solution and incubated at 37°C for 30 min in the dark. After staining, the cells were rinsed with 1X assay buffer and spun down in a centrifuge at 3500 rpm for 5 min. The supernatant was removed, and the cells were resuspended in 1X assay buffer. The cells were then put into FACS tubes and examined using an LSRFortessaTM Cell Analyzer (BD Biosciences). Using BD FACS DIVA™ software, the proportion of labeled cells was quantified from 10,000 cells per sample. FlowJo software version 9/10 was used to analyze the flow cytometry data.
Cytosolic And Mitochondrial Fractions
The isolation of the cytosolic and mitochondrial fractions was carried out using the Mitochondria/Cytosol Fractionation Kit (from abcam, Cambridge, UK). The procedure involved washing the cells with ice-cold PBS, spinning them down, and then resuspending the cell pellet in a mixture of 1X cytosol extraction buffer, protease inhibitor, and DTT for 10 min on ice. After centrifugation at 4°C for 15 min at 13,000 rpm, cytosolic proteins were collected and the pellets were resuspended in mitochondrial extraction buffer. The supernatant containing the mitochondrial proteins was then obtained by another round of centrifugation.
Make Of Mcl-1 Over-expression Vector And Transient Transfection
The pcDNA3.1-Mcl-1 over-expression vector was generated according to previously published methods [32]. HSC3 and HSC4 cells were treated with 1 µg of the blank pcDNA3.1 vector or the pcDNA3.1-Mcl-1 vector using Lipofectamine® 2000 (manufactured by Thermo Fisher Scientific, Inc.) as per the manufacturer's guidelines.
Statistical analysis
To ascertain statistical significance for intergroup comparisons, a two-tailed Student's t test was performed. To evaluate the significance of differences between the control and treatment groups, multiple group comparisons were analyzed using a one-way ANOVA with Tukey's post-hoc test. All graphs reflect the means and standard deviations of three separate studies. At *p < 0.05, statistical differences were regarded as significant.