Chemicals and antibodies
A caspase-3 assay kit ((Sigma SCP0084)), anti-β-actin (A2547), anti-rabbit-secondary antibody (Sigma A0545), and anti-mouse-secondary antibody (Sigma A9044) were purchased from Sigma (St. Louis, MO, USA). Resveratrol was kindly given by Chongqing Kerui Nanhai Pharmaceutical Company and a 500 mM stock solution was made in DMSO (0.1% v/v final concentration) stored at − 80 °C. Antibody against phospho-LKB-1 (sc-271,934), Bcl-2 (sc-492), AMPK (sc-19,128), phospho-AMPK (sc-101,630), Bax (sc-6236), and Beclin-1 (sc-11,427) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Compound C (sc-200,689) and Z-DEVD-FMK (sc-311,558) were also purchased from Santa Cruz Biotechnology Inc. Antibody against cleaved caspase-3 was purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Bid (bs-1153R) were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China). RPMI 1640 medium was obtained from Gibco and fetal bovine serum was purchased from Shanghai Excell Biological Technology Co., Ltd. (Shanghai, China). Annexin V, propidium iodide (PI) and the caspase-3 inhibitor Ac-DEVD-CHO (C1206) were obtained from the Beyotime Institute of Biotechnology (Jiangsu, China).
Cell lines and treatments
The human promyelocytic leukemia cell line HL-60 (ATCC® CCL240™) was purchased from the American Type Culture Collection (ATCC, USA). HL-60 cells were commonly cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum containing 100 U/ml penicillin and 100 U/ml streptomycin at 37 °C in a humidified atmosphere with 5% CO2. All experiments were performed only when the cells were growing during the exponential phase.
Cell proliferation assay
7 × 103 of exponentially growing cells were seeded in 96-well plates, and RSV of different concentration of 12.5–100 μM was added 24 h later. After incubation for 24 or 48 h, 20 μl of 1 mg/ml final concentration MTT(3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) was added, followed by a 4 h incubation at 37 °C, then 100 μl SDS-triplex liquid (10% SDS/5% isopropanol/12 mM HCl) was added to each well. The plate was then shaken for 10 min in the dark, then the absorbance value at 492 nm was read using a Micro-plate Auto-Reader (Labsystems). The percentage of cell viability was calculated as follows: cell viability (%) = (OD treatment/OD control) × 100% [10, 19].
Flow cytometry analysis
An annexin V-PI staining kit (Santa Cruz) was used to analyze the apoptosis using flow cytometry according to the manufacturer’s manual and our protocol published before . Briefly, cells were collected and resuspended in PBS after treatment, then annexin V-FITC and PI were added into the binding buffer. The log fluorescence values of annexin V-FITC (518 nm, FL1) and PI (620 nm, FL2) were shown on the X and Y axis, respectively.
Caspase-3 activity was analyzed using a commercial caspase-3 assay kit according to the our previous work . HL-60 cells were harvested and washed twice with ice-cold PBS after treatment. Cells were placed on ice for 15 min after being lysed by addition of 100 μl lysis buffer. Lysates were centrifuged at 15,000 g for 15 min followed by the protein concentrations assay using Bradford method.
Western blot analysis
Cells were harvested, washed with cold PBS and the cells pellet was lysed using ice-cold RIPA (50 mM Tris–HCL pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 5 mM aprotinin, leupeptin and pepstatin, 10 mM sodium orthovanadate and sodium fluoride) for 30 min. 50 μg protein was loaded and separated by SDS-PAGE on a 10 or 12% polyacrylamide gel. Proteins were transferred onto a nitrocellulose membrane and blocked with 5% non-fat milk in TBST buffer (50 mM Tris–HCl, pH 7.4, 0.15 mM NaCl, 0.1% Tween 20) for 30 min at room temperature. Blot membranes (Millipore, Bedford, MA, USA) were incubated (gently shaking) with 1st antibodies overnight at 4 °C. Blots were washed with TBST three times, 10 min each wash and then incubated with a 1:10,000 dilution of horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, the blot membranes were visualized with a SuperSignal West Dura detection kit (Pierce, Rockford, IL, USA) and exposed to medical Blue X-ray film after washed 3 times in TBST as we described previously .
RNA extraction and PCR
Total RNA was extracted from HL-60 cells with Trizol reagent (Invitrogen) and reversely transcribed using a Primescript® RT reagent kit purchased from Takara (DRR037A) as described previouly . PCR was performed using primers designed by Primer Premier 5.0 (Premier, Canada). The primers sequences were listed as follows: Beclin-1,5’-AGGTTGAGAAAGGCGAGACA-3′ and 5’-GCTTTTGTCCACTGCTCCTC-3′; the P62: 5’-AGCGTCAGGAAGGTGCCATT-3′ and 5’-TTCTCAAGCCCCATGTTGCAC-3′, and data were normalized using GAPDH transcript levels as internal control(GAPDH primers 5’-GGCCTCCAAGGAGTAAGACC-3′ and 5′- AGGGGAGATTCAGTGTGGTG-3′.) The amplified PCR products were separated by 2.0% agarose gel electrophoresis .
Immunofluorescence staining and microscopy
It is referred as to our previously published work . Briefly, Cells were collected and fixed with 4% formaldehyde at room temperature for 10 min. After rinsing with ice-cold phosphate-buffered saline (PBS) for three times, cells were permeabilized with 0.1% Triton X-100 for 10 min, and then blocked with 10% bovine serum albumin (BSA) for 1 h at room temperature. Cells were stained with a 1:500 dilution of corresponding primary antibodies overnight at 4 °C and incubated with CY3-conjugated anti-rabbit secondary antibody for 1 h at RT in the dark after washing with PBS for three times. DAPI was used to counterstain the nucleus and then mounted in glycerol followed by observing using a fluorescence microscope (Zeiss, Germany).
Fluorescence intensity analysis
For quantitative analysis of fluorescence intensity, cells treated with various concentrations of RSV for the indicated time were stained with fluorescence dye. Subsequently, equal numbers of cells were transferred to black 96-well culture plates and then assayed with a Multiskan Spectrum (Molecular Devices, SpectraMax M2e 200–100) at a 507 nm excitation wavelength and 529 nm emission wavelength for Rho123. The relative fluorescence intensity of all treated group was calculated supposing the control group as 100%.
For siRNA interference, cells were transfected using serum and antibiotics free culture medium containing Lipofectamine 2000 according to the manufacturer’s instructions and our protocol previously published . Briefly, the target sequences were 5′-GGAGCCAUUUAUUGAAACUTT-3′ (sense) and 5′-AGUUUCAAUAAAUGGCUCCTT-3′ (antisense) for Beclin1, 5′-GUGACGAGGAAUUGACAAUTT-3′ (sense) and 5′-AUUGUCAAUUCCUCGUCACTT-3′ (antisense) for P62. 5′-GACGUUGGUAACUGACAAATT-3′ (sense), 5′-AGUUUCAAUAAAUGGCUCCTT-3′ (antisense) for ATG5, 5′-GCCCUCUACUGAUUGUUAATT-3′ (sense) and 5′- UUAACAAUCAGUAGAGGGCTT-3′ (antisense) for LC3. Cells were directly used for follow-up experiments 24 h later after transfection.
Statistical analysis was performed using SPSS 15.0 software (SPSS Inc., Chicago, IL, USA). Data are expressed as means ± SD. Student’s unpaired t test was used to access differences between two values. A p-value of < 0.05 was considered to be statistically significant.