Cell cultures
The non-small cell lung cancer (NSCLC) cell line A549, pancreatic cancer cell line PANC and human normal hepatocyte line MIHA were obtained from the Cell Resource Center of Peking Union Medical College. Cells were cultured in DMEM (Gibco, USA) at 37 °C under 5% CO2 supplemented with 10% fetal calf serum (Gibco, USA) and 100 U/mL penicillin and streptomycin (HyClone, USA).
Reagents and antibodies
TMPyP4, 5,10,15,20-tetrakis(1-methylpyridinium-4-yl) porphyrin tetra(p-toluenesulfonate) and H2O2 were purchased from Sigma and dissolved in water. Antibodies against BAX, Bcl2, caspase 3, cleaved caspase 3, JNK, p-JNK, γ-H2AX, β-actin and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (USA). 53BP1 was from EMD Millipore (USA). β-actin was purchased from Proteintech (USA).
Cell viability assay
A549, PANC and MIHA cells were seeded on 96-well plates (3 × 103/well) and then treated with or without 10 μM TMPyP4 for 70 h, later treating them with 800 μM H2O2 for 2 h, after which 20 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (0.5 mg/ml) was added to each well for another 4h. The formazan reaction product was dissolved in DMSO (100 μl) after discarding the culture medium. Cell viability was detected by reading the absorbance at 490 nm with a Multiskan FC automatic microplate reader (Thermo Fisher, USA).
Measurement of intracellular ROS production
ROS were monitored using a ROS kit (Sigma, USA) according to the manufacturer’s instructions. In short, 1.5 × 105 cells were grown in six-well cell culture plates and incubated overnight. After treatment with or without drugs, cells were incubated with 10 μM DCFH-DA for 30 min at 37 °C in the dark. DCF fluorescence intensity was measured by flow cytometry (BD FACS Calibur, BD Biosciences, USA) and Nikon fluorescence microscopy (Nikon, Japan).
Colony formation
A549 and PANC cells were plated into 12-well plates (1000 cells/well) and incubated overnight until the cells attached to the dish and then were exposed to drugs or not. When sufficiently large colonies formed, the cells were washed, fixed with 4% paraformaldehyde and stained with crystal violet. Colonies in each plate were photographed under a light microscope and then the crystals were dissolved in 500 μL acetic acid (33%) and their absorbance was detected at 560 nm by a spectrophotometer.
Apoptosis assay
A549 and PANC cells were seeded in 6 cm2 dishes (1.5 × 105/well), incubated for 6h until the cells attached to the dish, and then treated with the indicated concentration of drugs. Cells were harvested for the Annexin V/PI apoptosis assay according to the manufacturer’s instructions and were analyzed by a flow cytometer.
Measurement of mitochondrial membrane potential (Δψm)
The cell mitochondrial membrane potential was detected by flow cytometry utilizing JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi-dazolylcarbocyanine iodide) staining. Briefly, A549 and PANC cells were treated with the indicated drug for the indicated times, harvested, and loaded with JC-1 solution for 20 min at 37 °C in the dark. Red fluorescence and green fluorescence intensity were determined by flow cytometry.
Immunofluorescence (IF) assays
Immunofluorescence (IF) assays were performed as previously described [21]. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 30 min, blocked with 5% GS for 1h at 37 °C, and incubated with primary antibodies (53BP1 or cleaved caspase 3) overnight at 4 °C in a humidified chamber. The next day, the cells were incubated with secondary antibody (anti-rabbit 488) for 1.5h at room temperature and mounted with DAPI. Fluorescent images were captured with a Nikon fluorescence microscope.
Comet assays
DNA damage was measured by neutral or alkaline comet assay [22]. In short, cells were treated with or without drugs, collected, and mixed with 0.5% low-melting-temperature agarose before being transferred onto slides, which were coated with 1.5% normal agarose. Then, the slides were lysed in 10 mM Tris (pH 8.0), 3% DMSO, 2.5 M NaCl, 0.5% Triton X-100, 1% N-lauroylsarcosine, and 100 mM EDTA and electrophoresed in 1% DMSO, 100 mM Tris-HCl, 300 mM sodium acetate at 1.5 V/cm for 25 min, mounted with 0.02 mg/mL PI solution and visualized under a Nikon fluorescence microscope. The analysis was performed with CASP.
Western blot analysis
The total protein was extracted and boiled for 10 min. The concentration of protein was detected by a Bradford assay, and the proteins were separated via SDS-PAGE and transferred to 0.25 μm PVDF membranes. The membranes were blocked with 5% skim milk (BD, USA) for 2 h at room temperature, incubated with primary antibodies at 4 °C overnight, washed with TBST three times, incubated with HRP-conjugated secondary antibodies for 1h at room temperature and detected using a Westar Supernova kit (Cyanagen).
Statistical analysis
GraphPad Prism 5 was used for statistical analysis. In histograms, all data are presented as the mean ± SD of at least three independent replicates for each experiment. The statistical significance of the data was assessed by Student’s two-tailed unpaired t-test or two-way ANOVA (*p < 0.05; **p < 0.01; ***p < 0.001).