Reagents and antibodies
Fisetin and z-VAD were purchased from Sigma, USA. The primary antibodies used in this study for western blotting were as follows: anti-HMGB1(Invitrogen, NY, USA), 1:1000 dilution; anti-ZBP1 (R&D system, USA), 1:1000 dilution; anti-RIP3(Calbiochem, San Diego, CA), 1:1000 dilution; anti-RIP1(Calbiochem, San Diego, CA), 1:1000 dilution; anti-MLKL (BD Biosciences, USA), 1:1000 dilution; anti-GAPDH (Santa Cruz, CA, USA), 1:5000 dilution.
Human ovarian carcinoma (OC) cell lines was obtained from Procell (Procell, China) and cultured in DMEM medium (Hyclone, USA) in which 10% fetal bovine serum (Gibico, USA) and 1% penicillin-streptomycin (Sigma, USA) were supplemented. Both cell lines were cultured in a 37% with 5% CO2 atmosphere. Fisetin was dissolved in dimethyl sulfoxide (DMSO) to a series concentration of 0, 25, 50 or 100 µmol/L then added into the culture medium to treat both cell lines for 72hr. v-ZAD (dissolved into 30 µmol/L concentration) was added into 100µmol/L fisetin treated cells for 72 hr to inhibit the apoptotic process.
Cell Viability Assay
The effect of fisetin on cell viability was examined by the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Briefly, A2780 and VOCAR-3 cells in both treated and control group were seeded in 96-well plates at the concentration of 75µmol/L cells per well. Cells were then incubated for 12h following fisetin treatment with a series of concentrations as mentioned. After incubation for 72h, MTT was added into each well following 4h incubation. The medium was then washed for 3 times to remove MTT. After stabilizing the blue formazan crystals by adding 150 µL of DMSO, the absorbance at 570 nm was detected with a microplate reader (Bio-Tek Instruments, VT) to determine the concentration of blue crystals.
Cell death analysis
Annexin V-FITC apoptosis detection kit (Sigma-Aldrich) was used for the detection of apoptotic cells. Cells were double-stained with PI and Annexin-V with a binding buffer for 30min at room temperature (RT). Apoptotic and necroptotic cells were analyzed with flow cytometry.
Transfection of ZBP1 siRNA
ZBP1 siRNAs (Santa Cruz Biotechnology, Santa Cruz, CA) were introduced to block the endogenous ZBP1 gene expression. Briefly, the ZBP1 siRNA (Thermal fisher, USA) was transfected into A2780 cells using siRNA transfection kit (Santa Cruz Biotechnology, Santa Cruz, CA) and following manufacturer’s instructions. After 24 hr transfection, fisetin + z-VAD treatment was then followed. The levels of ZBP1 protein in both cell lines were analyzed by Western blotting.
In vitro invasion assay
An in vitro cell invasion assay was introduced to measure the migration ability of both ZBP knockdown (ZBP1-/-) and control cells after fisetin + z-VAD treatment using the Matrigel-coated Transwell chamber (Corning, MA, United States). Briefly, both types of cells at a density of 24 cells/well were seeded onto the upper chambers in a serum-free medium with fisetin + z-VAD, while DMEM medium containing 10% FBS and 1% penicillin-streptomycin was filled into the lower chamber. After incubation for 24 h, the cells on top surface were removed while cells on the lower surface of the membranes were fixed with 4% paraformaldehyde for 10 min and stained using 0.25% crystal violet for 15 min. The invaded cells were introduced to an MTT assay.
Cell mitochondria isolation
Cell mitochondria isolation was performed using a Cell Mitochondria Isolation Kit (Thermal fisher, USA) according to the manufacturer’s instructions. Briefly, the cells were washed with PBS and digested with trypsin, then the cells were centrifuged at a speed of 100–200 g for 5 min to collect alkalotic cells. We then transferred the cells to clean tubes and homogenized the cells 10–30 times. The cell homogenate was centrifuged at a speed of 600 g at 4°C for 10 min. We transferred the supernatant to another clean tube and centrifuged it at a speed of 11000 g at 4°C for 10 min. The supernatant was cytosol, and the deposit was mitochondria.
RNA was extracted from isolated mitochondria by the PureLink RNA mini kit (Thermo Fisher Scientific, USA). The quantity and quality of extracted RNA were analyzed by using NanoDrop 2000 Spectrophotometer. Extracted RNA was prepared to synthesize cDNA according to the Quantinova reverse transcription kit. The Quantinova SYBR Green PCR Kit was used to determine mRNA expression on an Applied Biosystems PCR machine (95℃ for 10 min, 40 cycles of 95℃ for 15 s and 60℃ for 30 s, and final extension step of 60℃ for 30 s). Sequence of the primers used in the qRT-PCR experiment was listed as followed:
Cyt-c forward primer: 5’-CTG GGTGACGAGTGAAACTG-3’; reverse primer: 5’-TGAGCACAACAGGAACTGGA-3’; primer length in bp: 104bp.
GAPDH forward primer: 5’-GAAATCCCATCACCATCTTCCAGG-3’; reverse primer: 5’-GAGCCCCAGCCTTCTCCATG-3’; primer length in bp: 120bp.
Western blot analysis
Cells were cultured in absence or presence of respective intervention, then harvested and lysed in RIPA buffer for collecting the total protein. After iced incubation for 30 min, lysates were centrifugated at 12000 g for 5 min to separate the supernatants. The concentration of protein containing in the supernatants were measured by the BCA kit according to the manufacturer’s instructions. Next, 30 µg denatured protein samples were separated on SDS-PAGE gels and then transferred to PVDF membranes (Bio-Rad). After incubation of the membranes with primary antibodies at 4°C overnight, the samples were incubated with the secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature. The blots were visualized using a Leica Imaging System.
The statistical analysis was completed with the software package SPSS 23.0(SPSS Inc.; Chicago, IL, USA) and GraphPad Prism 6 (San Diego, CA) software. Data were presented as mean ± standard deviation. The Student’s t-test was used to compare the means of groups and P-values ≤ 0.05 were considered significant.