Reagents and Antibodies
N-Acetyl-L-cysteine (NAC), Z-VAD-FMK (caspase inhibitor) were purchased from Beyotime (Shanghai, China). SP600125 (JNK inhibitor) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The primary antibodies BAP31, Bcl-2, Bax, caspase 3, ERK, phospho-ERK, JNK, phospho-JNK, p38, phospho-p38, Cyclin E1, CDK2, p21 were purchased from Cell Signaling Technology (Dancers, MA). The primary antibodies HA, β-actin, PARP, Cyclin A2, AIF were purchased from Beyotime (Shanghai, China). The primary antibodies TOM20, Histone H3 were purchased from Absin Bioscience Inc (Shanghai, China).
Cell Culture
The human colorectal carcinoma cell line HCT116, human non-small cell lung cancer cell line A549, human bladder transitional cell carcinoma UM-UC-3, human hepatocarcinoma cell line HepG2, human gastric cancer cell line BGC823, human embryonic kidney cell line 293T were obtained from the Chinese Academy of Science (Shanghai, China). All the above cell lines were cultured in DMEM (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin-streptomycin (Gibco) at 37℃ in a 5% CO2 incubator (Thermo Fisher Scientific, Waltham, MA, USA).
Plasmid Construction and Transfection
The pcDNA3.1(-) plasmid (Mock) were obtained from Thermo Fisher Scientific (Waltham, MA, USA), and the pEGFP-N1 vector were obtained from TaKaRa (Beijing, China). Standard PCR techniques were used to generate cDNA encoding p20BAP31 (aa 1–164 of human BAP31) with a COOH-terminal HA tag that was cloned into the EcoRI and BamHI sites of pcDNA3.1(-) vector and pEGFP-N1 vector (PCR primer sequences are shown in Table S1). Cells were transiently transfected using Lipo8000™ transfection reagent (Beyotime, Shanghai, China). Briefly, 3\(\times\)105 cells were plated in 6-well plates the day before transfection. The plasmid: Lipofectamine ratio was 1:1.5. 2µg of plasmids were mixed in 125 µL of Opti-MEM, and then added 3µL Lipo8000™ transfection reagent, the solution was mixed gently and incubated at room temperature for 15 min. Then, the solutions were added to the plate drop by drop. Six hours later, the medium was replaced with fresh medium, and the cells were harvested at appropriate times.
Cell Proliferation Assay
The mock and p20BAP31 groups were transiently transfected as the above described the day after seeded in 96-well plates (3000 cells per well), the control group was directly seeded in 96-well plates (3000 cells per well), five multiple wells per group, and the plate was incubated at 37℃ in a 5% CO2 incubator. After each treatment period (0, 24, 48 and 72 hours), 10 µL of CCK-8 (Cell Counting Kit-8) solution (Beyotime, Shanghai, China) was added to each well, and the plate was incubated for 2 h. The optical density (OD) of the samples was measured by Synergy H1 microplate reader (Biotek, USA) at a wavelength of 450 nm.
Cell Apoptosis Analysis
Cells (3\(\times\)105 per well) were seeded in 6-well plates, harvested after 48 h of transfection, washed repeatedly with cold phosphate-buffered saline (PBS), and resuspended in 195 µL of binding buffer. Annexin V-FITC reagent (4 µL) and PI (8 µL, Beyotime) were added to the samples and mixed gently. After 15 min incubation in the dark at room temperature, the stained cells were analyzed immediately using a FACScan flow cytometry (BD Biosciences, San Jose, CA, USA) and analyzed with the Annexin V-FITC/PI apoptosis method.
Western Blot Analysis
Cells were lysed in RIPA buffer (Beyotime, Shanghai, China) containing 1% protease inhibitor cocktail and PMSF (1 mM, Sigma-Aldrich, St. Louis, MO, USA) for 30 min. After centrifugation at 12,000×g for 15 min, the supernatant was collected. The mitochondria proteins were isolated and lysed by using Cell Mitochondria Isolation Kit (Beyotime). The intranuclear and extranuclear proteins were extracted using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) according to the manufacturer's protocol. The protein concentration was determined by the BCA method. BCA kits were purchased from Beyotime. After determination of the protein concentrations, the lysates were mixed with the 5×loading buffer and denatured by boiling for 10 min. The lysates with the equal amount of proteins were separated on 10–15% SDS-PAGE, and then transferred to PVDF membrane (Merk Millipore, Darmstadt, Germany). After blocking with 5% skimmed milk at room temperature for 0.5 h, the PVDF membrane was incubated with corresponding antibodies at 4℃ overnight and then with HRP conjugated secondary antibodies at room temperature for 1 h. Finally, the protein bands were visualized with Bio-Rad ChemiDoc TM imaging systems using an ECL detection kit (Thermo Fisher Scientific, Waltham, MA, USA).
Hoechst Staining
HCT116 cells (5\(\times\)104 per well) were seeded in 24-well plates and transfected for 48 h. Afterwards, the cells were washed three times with phosphate-buffered saline (PBS) and incubated with Hoechst 33342 (Beyotime, 2 µg/mL in medium) at room temperature for 5 min in the dark. The nuclear images were observed with a DMI3000B fluorescence microscope (Leica, Germany), the changes in nuclear morphology were evaluated and photographed.
Colony-Forming Assay
HCT116 cells were seeded into 6-well plates at a density of 2000 cells per well and transfected with plasmid. All conditions were performed in triplicate using untreated cells as reference control. Cells were cultivated for 7 days, at which point they were fixed with 4% formaldehyde for 30 min, stained with 0.1% crystal violet for 15 min. Finally, the plates were washed with water and air-dried at RT. The colony counts were determined using the ImageJ software (v. 1.8.0) program (NIH, Bethesda, MD, USA).
Wound-Healing Assay
The migration of HCT116 cells was evaluated in wound-healing assays. In brief, when cells grew to 90% confluence in 6-well dishes, a “wound” was created by scratching the monolayer with a 200 µL pipette tip. Following transfected with plasmid, the cells were examined by a DMI3000B fluorescence microscope (Leica, Germany) at time 0, 12, and 24 h.
Mitochondrial Membrane Potential (MMP) Assay
MMP was examined using a JC-1 mitochondrial membrane potential assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, HCT116 cells (3\(\times\)105 per well) were plated in 6-well plates. After transfection with plasmid for 48 h, cells were collected and mixed with 1 mL of 1×JC-1 working solution at 37℃ in the dark for 20 min. Subsequently, the cells were centrifuged (1000 rpm, 5 min) and washed twice by 1×JC-1 staining buffer. The collected cells were resuspended in 500 µL of JC-1 staining buffer and immediately analyzed by a FACScan flow cytometry (BD Biosciences, San Jose, CA, USA).
Moreover, MMP was also examined by fluorescence microscope. HCT116 cells (5\(\times\)104 per well) were plated in 24-well plates. After transfection with plasmid for 48 h, the cells were washed once with PBS. Afterwards, 0.5 mL of the cell culture solution and 0.5 mL of JC-1 working solution were successively added, thoroughly mixed, and incubated at 37°C for 20 min in the dark. The cells were washed twice with 1×JC-1 staining buffer and finally added fresh culture medium. The images were observed with DMI3000B fluorescence microscope (Leica, Germany).
Measurement of Intracellular ROS Levels
ROS levels were quantified in accordance with the protocol of the Reactive Oxygen Species Assay Kit (Beyotime, Shanghai, China). The Reactive Oxygen Species Assay Kit allows for the quantitative measurement of ROS, namely superoxide radicals in cells undergoing oxidative stress. HCT116 cells (3\(\times\)105 per well) were plated in 6-well plates. After transfection with plasmid for 48 h, the cells were stained with DCFH-DA (10 µM) at 37℃ for 20 min in the dark and then washed with serum-free DMEM three times. Finally, cells were collected for ROS analysis using a FACScan flow cytometry (BD Biosciences, San Jose, CA, USA).
In addition, ROS levels were also examined by fluorescence microscope. HCT116 cells (5\(\times\)104 per well) were seeded into 24-well plates. After transfection with plasmid for 48 h, the cells were stained with DCFH-DA (10 µM) at 37℃ for 20 min in the dark and then washed with serum-free DMEM three times. Finally, the images were observed with DMI3000B fluorescence microscope (Leica, Germany).
Cell Cycle Analysis
For the cell cycle distribution analysis, HCT116 cells (3\(\times\)105 per well) were plated in 6-well plates. After transfection with plasmid for 48 h, the cells were collected with the growth medium, centrifuged, washed with cold PBS and fixed with 70% ice-cold ethanol overnight at -20℃. The next day, the cells were washed twice with cold PBS, gently resuspended in 500 µL of propidium iodide (PI) working fluid (staining buffer: PI (20×): RNAase (50×) = 100: 5: 2) (Meilun Biotechnology Co. Ltd., Dalian, China), and incubated at 37℃ for 30 min in the dark. Cell cycle analysis was performed using a FACScan flow cytometry (BD Biosciences, San Jose, CA, USA).
Immunofluorescence Assay
5\(\times\)104 cells were plated in 24-well plates. After 48 h of transfection with plasmid, the cells were fixed in 4 paraformaldehyde for 30 min, permeabilized with 0.2% Triton X-100 for 30 min, blocked with 1 BSA-PBS for 30 min at room temperature, and then incubated with anti-AIF antibody (1:100 dilution) at 4°C overnight. The fluorescent secondary antibodies were incubated with the cells at room temperature for 2 h in the dark. The nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI, 300 nM in PBS) for 5 min, the images were captured using the DMI3000B fluorescence microscope (Leica, Germany).
Statistical Analysis
All results were presented as the mean ± standard error (SE) from at least three independent experiments. Curve fittings, data, and statistical analysis were carried out by the GraphPad Prism program (Prism 6, San Diego, CA, USA). Differences between groups were assessed by one-way analysis of variance (ANOVA). For all analyses P-values represent 2-sided tests of statistical significance effects. P-values < 0.05 was considered statistically significant. SPSS v.13.0 (IBM, NY, USA) was used for the calculation of the differences. Non-linear regression was fitted to the data points using GraphPad Prism program.