NCI-H460 (RRID:CVCL_0459, H460 for short) and H460-Cis cells were purchased from the Shanghai Cell Collection (Chinese Academy of Sciences). Both cell lines were maintained in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA). All media were supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA). H460-Cis cells are treated with 2 mg/ml cisplatin to maintain cisplatin resistance character. All human cell lines have been authenticated using STR profiling within the last three years has been included. Cells were maintained at 37°C in a humidified atmosphere with 5% CO2. Cells were regularly tested for mycoplasma and were mycoplasma free as previously reported (21).
APR-246 and MG132 were purchased from MedChemExpress (China). Cycloheximide (CHX) was purchase from MP Biomedicals (France). N-acetyl-L-cysteine (NAC) was bought from Beyotime Biotechnology (Beijing, China). Each reagent was dissolved in its favorable solvent liquid at appropriated concentration. All the reagents were stored at -20°C for long-term preservation.
Quantification of ROS level
ROS was assayed using 2',7'-dichlorofluorescin diacetate (DCFDA) cellular ROS detection assay kit (Beyotime Biotechnology, Beijing, China) following manufacturers’ protocol. In short, cells were seeded in 6-well plates. Then DCFDA was added to the medium 1:500 and incubated for 30 min then cells were harvested and analyzed using flow cytometry (BD Biosciences, Mississauga, Ontario). All experiments were performed in triple times.
Quantification of NADP+/NADPH
The intracellular NADP+ and NADPH levels were measured using the NADP+/NADPH Assay Kit (Abcam, Inc., MA, USA). Experiments were performed according to the manufacturer’s protocols, and the NADP+, and NADPH concentrations were determined colorimetrically based on absorbance at 565 nm. All experiments were performed in triple times.
Quantification of GSH and GSSG
Total (GS), oxidized (GSSG) and reduced (GSH) glutathione concentrations were measured using the GSH/GSSG Ratio Detection Assay Kit (Abcam, Inc., MA, USA) and a fluorescence microplate reader with excitation and emission wavelengths of 490 and 520 nm, respectively. All experiments were performed in triple times.
Same as before (22), cells at a concentration of 5 × 104 cells/well were seeded in 100 μl culture medium into microplates (tissue culture grade, 96 wells), and the microplates were incubated in cell cultures for 72 hours or 96 hours at 37°C and 5% CO2. After the incubation period, 10 μl of the MTT labeling reagents (final concentration 0.5 mg/ml) were added to each well at preset time points, including 0 hour, 24 hours, 48 hours and 72 hours. Then we incubated the microplate for 4 h in a humidified atmosphere. And 4 hours later, 100 μl of the solubilization solution was added into each well. After hatching in the incubator overnight, we checked for complete solubilization of the purple formazan crystals and measure the absorbance of the samples using a microplate reader. The wavelength to measure absorbance of the formazan product is between 570 nm.
Colony formation assays
Briefly, cells were trypsinized, suspended in complete medium, then counted and re-plated in six-well plate to allow formation of macroscopic colonies. Plates were incubated at 37°C for 7 to 20 days, fixed with methanol, stained with crystal violet, and colonies containing at least 50 cells in size were counted.
Apoptosis and cell cycle assay
In apoptosis assay, cells were classified into different groups, according to combination of APR-246 and/or NAC. Then nonadherent and adherent cells were collected at 48 hr after treatment. Apoptosis was determined using the Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit (Roche, USA) with FACS Calibur flow cytometer (FCM) (BD Biosciences, Mississauga, Ontario). For cell cycle analyses, cells were dyed with PI and detected with the FACS Calibur FCM.
Reverse-transcription PCR and real-time PCR assay
RNA extraction and RT-PCR were performed as described previously (23). Total RNA was extracted with TRIZOL reagents following the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Potential DNA contamination was removed by RNase-free DNase treatment (Promega, Madison, WI, USA). cDNA was prepared with the MMLV Reverse Transcriptase (Takara, Japan). Relative quantitation was determined using the ABI QuantStudio6Flex System (Thermo Fisher, USA). Primer sequences were provided in Supplemental Table 1. All the experiments were performed in triplicate.
Western blotting analysis were performed as described previously (24). Equal amounts of protein extracts were separated by SDS-polyacrylamide gel, transferred to a polyvinyl difluoride membrane (PVDF) membrane (Roche; Roche Diagnostics, Basel, Switzerland) at 37°C for 1 hour, followed by incubation with specific primary antibodies overnight at 4°C. Membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour and detected using the ECL kit (Thermo Scientific, Rockford, IL, USA). The detailed sources and other information of antibodies were listed in the supplemental materials (Supplemental Table 2).
CHX-mediated chase assay
To assess protein stability, cells were treated with 200 µg/mL CHX to stop de novo protein synthesis. At the preset time points, cell lysates were harvested and then subjected to immunoblotting.
Primers and antibody
The detailed information of primers and antibodies were documented in the supplementary information (Supplemental Table1 and Supplemental Table 2).
Female BALB/c nude mice (4~5 weeks of age) were housed in the Shandong Cancer Hospital and Institute according to protocols approved by the Shandong Cancer Hospital Animal Care Committee. For xenografts, about 6.0 × 106 viable cells in 100 μl PBS were injected subcutaneously into the nude mice. Five animals per group were used in each experiment. APR-246 (50mg/kg or 100mg/kg) were injected i.p. at preset timepoint. NAC (5 mg/mL) was given in the drinking water for the length of the experiment until sacrifice. Tumor sizes were measured every 4 days, and mice were euthanized when tumors reached 1.5 cm in diameter. The volume was calculated according to the formula: 1/2 × length × squared width. All studies were approved by the Animal Care Committee of Shandong Cancer Hospital.
Data from the two groups were evaluated statistically by a two-tailed unpaired t-test (GraphPad Software, San Diego, CA, USA). In these analyses, p values less than 0.05 were considered significant (*p < 0.05, **p < 0.01, ***p < 0.001).