Compound
TPL (molecular formula, C20H24O6) was purchased from Shanghai Tongtian Biotechnology Co., Ltd. The material was composed of white powder and 97% pure by HPLC determination.
Cell Culture
Human hepatocellular carcinoma cell line Huh7 was grown in Dulbecco's modified Eagle's medium (HyClone) supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 ug/ml streptomycin.
Analysis of Huh7 Cell Death induced by TPL and/or TNF-α
Recombinant human TNF-α was purchased from Peprotech. Death of Huh7 cells induced by TPL and/or TNF-α was analyzed by Cell counting kit 8 (CCK-8) (Dongren Chemical Technology Co., Ltd., Shanghai). Briefly, cells were seeded in a 96-well plate (3×103 cells/well) and then treated with different concentrations of TPL or TNF-α (0, 2.5, 5, 10, 20 ng/ml) or a combination of TPL (5 ng/ml) and the precedent concentrations of TNF-α for 48 h. Afterwards, all the treated cells were incubated with CCK-8 solution in the cell incubator for another 3 h. Then, a microplate reader (Bio-Rad, Hercules, CA) was used to measure the absorbance at 450 nm.
Besides, Huh7 cells were seeded in a 6-well plate at a density of 1.7×105 cells per well and cultured overnight. After the cells were attached, TPL and/or TNF-α was added in the culture medium to incubate cells for approximately 43 h, a time point at which death of the cells was visible. Thereafter, the cells were harvested for determination of the protein c-FLIP as well as proteins participating in apoptosis.
Treatments of Huh7 Cells for Analysis of FLIPS Downregulation promoted by TPL
Huh7 cells were maintained in mediums containing 0, 5, 10, 20, 25 ng/ml TPL for 24 h or 48 h before being harvested for determination of c-FLIP protein and mRNA. Besides, Huh7 cells were incubated with medium containing 20 ng/ml TPL for 0, 6, 12, 22, 32 h and then collected for evaluation of c-FLIP protein levels. Moreover, Huh7 cells were untreated or pre-treated with proteasome inhibitor Lactacystin (LC) (APExBIO Technology LLC, Houston) or MG132 (MedChemExpress, Shanghai) for 2 h before the addition of TPL, and 4 h, 8 h, 12 h (for cells pre-treated with LC) or 2 h, 4 h, 6 h (for cells pre-treated with MG132) after TPL was added in, cells were harvested for analysis of FLIPs levels.
Real-time PCR
Total RNA of the cell was extracted and purified with RNAiso Plus (TaKaRa, Otsu, Japan) and chloroform. Then cDNA was obtained from reverse transcription reaction by using PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa). Real-time PCR were carried out on ABI PRISM 7900HT/FAST (Applied Biosystems, Foster, CA) at 40 cycles of 95 ℃ for 5 s and 60 ℃ for 30 s. Primers for FLIPL were 5’-GGCTCCCCCTGCATCAC-3’ and 5’-TTTGGCTTCCCTGCTAGATAAGG-3’. Primers for FLIPs were 5’-ACCCTCACCTTGTTTCGGACTAT-3’ and 5’-TGAGGACACATCAGATTTATCCAAA-3’. Levels of Both isoforms were normalized to GAPDH and fold change in the level of each isoform between treated and control group was calculated with the 2-ΔΔCt method. GAPDH primers were: 5’-GGAGCGAGATCCCTCCAAAAT-3’
and 5’-GGCTGTTGTCATACTTCTCATGG-3’.
Western Blotting
The harvested cells were lysed in RIPA buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 0.5% deoxycholic acid, 1% Triton X-100) supplemented with 10% PMSF. Protein concentration of cell lysate was determined using BCA protein assay kit (Pierce). Equivalent amount of protein (50 ug) was fractionated by precast mini polyacrylamide gels (SurePAGETM, Bis-Tris, 4~20%) (GenScript, Nanjing, Jiangsu) and undergone western blotting. The proteins were visualized by enhanced chemiluminescence (Proteintech Group, Inc., Chicago, IL) according to the manufacturer’s instructions.
Determination of ROS Level
The cellular ROS was detected by fluorescent probe DCFH-DA (Beyotime, Shanghai, China). Briefly, cells were inoculated into 6 -cm dishes at a density of 1 x 106 cells/dish, incubated overnight, and then treated with 20 ng/ml of TPL for 24 h. After that, the culture medium was changed into 2 ml DMEM containing 10 uM of DCFH-DA and the cells were incubated for another 1 h in the incubator. Then the cells were washed 3 times with DMEM to fully remove the left DCFH-DA which did not enter the cell. Finally, fluorescence intensity in each cell was determined by the flow cytometer on which 488 nm was used as the excitation wavelength and 525 nm was used as the emission wavelength. Mean fluorescence intensity (MFI) representing the level of ROS was quantitated with Flowjo10 software.