2.1 Cell culture
Human colon cancer HT29 cell line (ATCC HTB-38) and Burkitt lymphoma Raji cell line (ATCC CCL-86) were cultured in RPMI-1640 media containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), 1% w/v glutamine and 1% w/v antibiotics. Both the cell lines were cultured at 37°C in a humidified incubator containing 5% CO2.
2.2 Reagents and antibodies
z-VAD-fmk, a cell-permeable and irreversible pan-caspase inhibitor and cisplatin were from MedChemExpress (Monmouth Junction, NJ, USA). TCN was obtained from Kunming Institute of Botany, the Chinese Academy of Sciences (purity >99%, HPLC analysis). Dimethyl sulphoxide (DMSO, Sigma) was used to dissolve TCN. The antibody against β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-RIP1 was from BD Biosciences (San Jose, CA, USA). Anti-RIP3 and anti-phosphorylated MLKL were from Abcam (Cambridge, MA, USA). The antibody against cleaved-Caspase 3 and Ki67 was obtained from Cell Signaling Technologies (Danvers, MA, USA).
2.3 Cell viability assay
The cell proliferation rate was assessed by plating cells into 96-well plates (2 × 103 cells per well) and absorption measurement was performed at 490 nm by a BioTek microplate reader (Beckman, Brea, CA, USA) with CCK-8 kit(Beyotime, China).
2.4 Cell permeability assay
Cell permeability assay was performed using the cell-impermeable dye Sytox Green (Invitrogen, USA). Briefly, cells were incubated with 30nM Sytox Green dead cell stain for 10 min in the dark at room temperature and then imaged with a DMI3000 fluorescence microscope (Leica, Germany).
2.5 Transmission electron microscopy (TEM)
TEM was performed to identify the cells undergoing necroptosis. Cells were fixed with ice-cold 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) at 4°C for 4 h, then were post-fixed in 1% osmium tetroxide, dehydrated in graded alcohol and embedded in epoxy resin. The sections were counterstained with uranyl acetate and lead citrate and detected with a HT7700 TEM (Hitachi, Japan) at an accelerating voltage of 100 kV.
2.6 Immunofluorescence analysis
The cells were grown on glass coverslips overnight and treated with DMSO or TCN for 24 h, followed by fixation with 4% paraformaldehyde. After permeabilization with 0.3% Triton X-100 in PBS, the cells were blocked with 5% BSA for 1 h and then incubated with the primary antibody overnight. The coverslips were washed with PBS and then incubated with appropriate fluorescent secondary antibody for 1 h, followed by washing and mounting using DAPI. Images were obtained by confocal microscopy (TCS SP8, Leica) and quantified using the NIH Image J software.
2.7 Measurement of the cellular oxygen consumption rate using the XF extracellular flux analyzer
Cells were plated in XF96 cell culture plates (Seahorse Bioscience, North Billerica, MA, USA) and incubated overnight. Cells were equilibrated with Seahorse XF base medium supplemented with glucose, glutamine, pyruvate and incubated into a 37 °C non-CO2 incubator for 45 min to 1 h prior to the XF assay. Oligomycin was prepared for final concentrations of 1.5 μM, Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), rotenone and antimycin A were prepared for a final concentration of 0.5 μM, and they were sequentially injected from the reagent ports automatically to the wells. Real-time measurements of oxygen consumption rate (OCR) in pmole per min for cells in culture medium were plotted over time. The OCR measurements were normalized to protein levels in each well.
2.8 Measurement of mitochondrial ROS accumulation
Cells were plated into 6-well plates and stained with 5 μM MitoSOX Red (Molecular Probes, Eugene, OR, USA) for 30 min at 37 °C. Mitochondrial ROS content was detected by flow cytometry (Becton Dickinson, USA) and analyzed by flowjo ver.7.6 software. The fluorescence intensity reflected the amount of mitochondrial ROS and an increase in fluorescence intensity represents an enhanced generation of mitochondrial ROS.
2.9 Immunohistochemical analysis
The tumor tissue sections were deparaffinized in environmentally friendly dewaxing agent (solarbio, china) and rehydrated with an ethanol-aqueous solutions of decreasing concentrations. For antigen retrieval, tissue sections were incubated in 10 mM sodium citrate buffer (pH = 6.0) for 20 min in a microwave oven. The endogenous peroxidase activity was removed by incubating with 3% hydrogen peroxide for 10 min and was blocked in normal donkey serum for 30 min. The primary antibodies (anti-RIP3, anti-Ki67) were applied at 4 °C overnight. Chromogen was developed using DAB (Zsgbbio, China) and counterstained with hematoxylin staining kit. Immunohistochemical staining of these sections was evaluated based on all of the available tumor cells or epithelial cells meeting the typical morphological criteria by 3 pathologists using the qualitative scale that is described in the literature.
2.10 Tumor xenograft studies
Xenograft experiment using HT29 cells was performed by subcutaneous injection of 3 × 106 cells into 5-week-old BALB/c nu/nu female mice. After the tumors grew to a volume of about 80–100 mm3, mice were randomly divided into four groups (n = 4 for each): untreated (vehicle), treatment with TCN (1 mg/kg) or DDP (1 mg/kg), treatment with TCN (0.5mg/kg) combined with DDP (1mg/kg) by intraperitoneal injection every day for 13 days, respectively. Tumor volume was calculated according to the formula (V = length x width2/2). At the end of experiments, the mice were euthanized by CO2 inhalation and the tumors were stripped and weighed. Animal care experimental procedures were conducted in accordance with the approval of Xiangya hospital of Central South University (Changsha, China).
2.11 Statistical analysis
Statistical analysis was performed using ANOVA and two-tailed Student‘s t test and a p value <0.05 was considered statistically significant. Statistical calculations were performed using SPSS ver.16.0 software.