Chemicals and cells
Cinobufagin was purchased from Yuanye Biotechnology (Shanghai, China). Stock solution of cinobufagin was prepared in 100% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) and then diluted in complete cell culture medium to make working solutions. The same solutions without cinobufagin were used as vehicle controls.
Human SW480, SW1116 colorectal adenocarcinoma and BEAS-2B bronchial epithelial cell lines were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and the human L-O2 liver and NCM460 colon epithelial cell lines were purchased from KenGen (Nanjing, China). All other cell lines were bought from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Life Technologies Corporation) containing 10 % fetal bovine serum (FBS, Kangyuan, China). The cells were maintained at 37°C in a humidified incubator with 5% CO2, following instructions from the providers. Cell authenticity was confirmed by short tandem repeats (STR) profiling.
MTT cell proliferation assay
Cells were seeded in triplicate wells of 96-well plates at 4 × 103 cells per well and treated with cinobufagin (drug concentrations and treatment times were indicated in the figures and figure legends). 20 μl of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) was added to each well, and the plates were kept at 37°C for another 4 h. After carefully removing the MTT-containing medium, 150 ml of DMSO was added to each well and the plates were kept at 37oC for 10 min with shaking. Absorbance was read at 595 nm by a BioRad 680 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). All MTT assays were repeated three times and the data were presented as mean + standard deviation (SD) of three independent experiments.
Measurement of cellular ROS
Cells were seeded in triplicate wells of 6-well plates at 1 × 105 cells per well and treated with cinobufagin. Cellular ROS were stained by a cell-based ROS assay kit (Beyotime, Shanghai, China) following manufacturer’s instructions. Briefly, cells were washed with PBS, and incubated with 10 μM (DCFH-DA) for 30 min at 37°C in the dark. After washing three times in PBS, photos were taken immediately using an Olympus fluorescent microscope. The cells were then collected through trypsinization and analyzed on the BD FACS-Calibur ﬂow cytometer (BD Biosciences, San Jose, CA, USA). Cellular ROS levels were expressed as the average 2’,7’-dichlorofluorescein fluorescence intensity. Results were mean + SD of three independent experiments.
Cells were seeded on round coverslips in 24-well plates and treated with cinobufagin. After washing in PBS, cells were fixed in ice-cold 4% paraformaldehyde (PFA) for 30 min and washed three times with phosphate buffer saline (PBS).
For immunostaining of 8-oxoguanine (8-oxoG), fixed cells were incubated in Alexa 488-conjugated avidin (Rockland Immunochemicals, Limerick, PA, USA) (0.5 mg/ml) for 1 h at room temperature, washed three times in PBS, and the coverslips with stained cells were sealed on glass slides in the VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Images were acquired using a Zeiss LCM 510 confocal microscope, signal intensity was quantified using the ImageJ software. At least 50 cells per sample were measured.
For immunofluorescent staining of 53BP1 or gH2AX, the cells were incubated sequentially in blocking buffer (3% fetal bovine serum, 0.1% Triton X-100 in PBS), rabbit anti-53BP1 (Bethyl Laboratories, Montgomery, TX, USA) or rabbit-anti-gH2AX-pS139 (Abcam, Cambridge, UK) primary antibody, and Cy3-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), each for 1 h at room temperature. Cells were then washed three times in PBS, and the coverslips with stained cells were sealed on glass slides in the VECTASHIELD Mounting Medium with DAPI (Vector Laboratories). Images were acquired using a Zeiss LCM 510 confocal microscope. Foci were quantified using the ImageJ software, more than 100 cells per sample were analyzed.
Measurement of DNA strand breaks in individual cells by the alkaline comet assay (single cell gel electrophoresis assay) was performed according to the instructions included in the OxiSelect Comet Assay kit (Cell Biolabs, San Diego, CA, USA). Cells grown in 6-well plates were treated with cinobufagin and collected through trypsinization. Cell pellets were resuspended in 1.2% low-melting point agarose maintained at 37°C at 10 x 105 cells/ml, which were then layered on a frosted slide from the OxiSelect Comet Assay kit. The slides were stored at 4°C overnight in pre-cooled lysis buffer (100 mM EDTA, 2.5 M NaCl, 10 mM Tris–HCl, 1% Triton X-100 and 10% DMSO, pH 10.0). After washing twice with an enzyme buffer (40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA and 0.2 mg/ml BSA, pH 8.0), the slides were incubated in the enzyme buffer with or without 1.0 mg/ml OGG1 (ProSpec, Rehovot, Israel) for 45 min at 37°C, washed briefly in the enzyme buffer, and denatured in pre-chilled alkali buffer (300 mM NaOH, 1 mM EDTA) in a horizontal electrophoresis chamber (Bio-Rad Laboratories) for 30 min. Electrophoresis was then proceeded at 20 V and 300 mA in the same buffer for 30 min. After incubation in cold neutralizing buffer (250 mM Tris–HCl, pH 7.5) for 30 min, slides were immersed in cold 70% ethanol for 5 min and allowed to air dry. At the end, cells were stained with Vista Green DNA dye provided by the kit at room temperature for 15 min. Images were acquired with an Olympus fluorescent microscope and quantified using the Comet Assay IV software (Perceptive Instruments, Edmunds, UK). The tail moment was defined as the product of the tail length and the fraction of total DNA in the tail, at least 50 cells per sample were analyzed.
Flow cytometric analysis of cell cycle and apoptosis
Cells were seeded in triplicate wells of 6-well plates at 1 × 105 cells per well, treated with cinobufagin, and collected through trypsinization. The cell pellets were washed twice in PBS before the following analyses. All experiments were performed three times and the results were presented as mean + SD.
For cell cycle analysis, cell pellets were fixed in 70% ice-cold ethanol at −20°C for 1 h. After washing in PBS twice, cells were stained with working solutions from the Cell Cycle Detection kit (BestBio, Shanghai, China). Samples were loaded onto and analyzed by the MoFlo XDP Cell Sorter (Beckman Coulter, Indianapolis, IN, USA). Data were processed with the FlowJo software (FlowJo, Ashland, OR, USA).
For the analysis of apoptosis, cells were resuspended in a binding buffer from the Annexin V-FITC Apoptosis Detection kit (BestBio) according to the instructions provided by the manufacturer. The cells were loaded onto and analyzed by the MoFlo XDP Cell Sorter (Beckman Coulter) and data were processed with the CytExpert software (Beckman Coulter).
Cells were seeded on round coverslips in 24-well plates and treated with cinobufagin. After washing in PBS, cells were fixed in ice-cold 4% paraformaldehyde (PFA) for 30 min and washed three times with PBS. Fixed cells were incubated in 0.1% Triton X-100 in PBS for 2 min on ice, washed twice with PBS, and then incubated in a working solution from the One-Step TUNEL Apoptosis Assay kit (Beyotime) for 60 min at 37°C. After washing three times in PBS, the coverslips with stained cells were sealed on glass slides in the VECTASHIELD Mounting Medium with DAPI (Vector Laboratories). Photos were captured using a Zeiss LCM 510 confocal microscope.
Measurement of MMP
Cells were seeded in triplicate wells of 6-well plates at 1 × 105 cells per well and treated with cinobufagin. Mitochondrial membrane potential (MMP) was measured using a commercial Mitochondrial Membrane Potential Assay kit with JC-1 (Beyotime) following manufacturer’s instructions. Images were taken using an Olympus fluorescent microscope.
Cells grown in 6-well plates were scraped off the plates in 100 ml of radioimmunoprecipitation buffer (150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 50 mM Tris, pH 8.0) (Sigma-Aldrich) with 1 mM phenylmethane sulfonylfluoride (Sigma-Aldrich). Samples were centrifuged at 4°C for 20 min at 12,000 x g and protein concentrations were determined by a BCA Protein Assay kit (Dingguo, Changchun, China). Proteins were denatured at 95°C for 10 min, separated on a 12% SDS-PAGE gel, and transferred to PVDF membranes. The membranes were blocked in 5% (w/v) non-fat milk in TBST (10 mM Tris, 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h at room temperature, and then incubated sequentially in primary and secondary antibodies each for 1 h at room temperature. Signals were developed using a Tanon-5200 chemiluminescence image analysis system (Tanon, Shanghai, China). All experiments were performed three times.
Primary antibodies included gH2AX-pS139 polyclonal antibody (ab11174) (Abcam, Cambridge, UK); Chk1-pS317 (12302S) and Chk2-pT68 (2661S) (Cell Signaling, Danvers, MA, USA); Chk1 (bs-1681R), GAPDH (bs-2188R), CDC25C (bs-10579R), activated caspase 3 (bsm-33199M), caspase 3 (bsm-52289R), CDC25C-pS216 (bs-3096R), p53-pS15 (bs-3702R) and p53 (bs-2092R) (Bioss, Beijing, China); CDC25A (abs131784) and Chk2 (abs131635) (Absin Bioscience, Shanghai, China); 53BP1 (A300-272A) (Bethyl, Montgomery, TX, USA). Secondary antibodies were goat anti-mouse-Alexa 488 (115-545-003) and goat anti-rabbit-Cy3 (111-165-003) (Jackson ImmunoResearch, West Grove, PA, USA); goat anti-mouse-HRP (bs-40296G-HRP) and goat anti-rabbit-HRP (bs-40295G-HRP) (Bioss).
Statistical analyses were carried out in GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). Comparisons between two groups were performed by unpaired two‑tailed Student's t‑test, and p < 0.05 was considered statistically significant.