JS-K (Fig.1A), PP2A-Cα (siRNA) and control siRNA were available from Santa Cruz Biotechnology (San Diego, CA). Carboxy-PTIO was obtained from Beyotime Institute of Biotechnology (Shanghai, China). Annexin V-FITC/PI kit was from BD Biosciences (NJ, San Diego, CA, USA). Apoptosis Antibody Sampler Kit, antibodies for phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), p44/42 MAPK (Erk1/2), Bax, Bcl-2, CIP2A, phospho-c-Raf (Ser338), phospho-MEK1/2 (Ser217/221), phospho-c-Myc (Ser62), phospho-c-Fos (Ser32) and phospho-Elk-1 (Ser383) mouse mAb were purchased from Cell Signaling Technology (Beverly, MA, USA). The dilution ratio of CST antibodies is 1:1000. β-actin (1:5000), Histone H3, HRP-conjugated affinipure goat anti-rabbit IgG, and HRP-conjugated affinipure goat anti-mouse IgG were supplied from Proteintech Group, Inc (Wuhan, China). The methylthiazolyl tetrazolium biomide (MTT) was from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). CIP2A siRNA, CIP2A cDNA, PPP2CA cDNA (PP2A-Cα, GV230 carrier, NheI/AgeI enzymatic cutting) was obtained from Shanghai Genechem Co., Ltd.
Cell proliferation determination
HepG2 cells were seeded into 96-well cell plates at a final density of 1.0×104 cells/well for 12 h and incubated with different concentrations of JS-K for a specific time. Then, MTT at a concentration of 5 mg/mL was added to each well and incubated for another 4 h. DMSO was used to dissolve the formazan crystals. Finally, the absorbance was measured using a microplate reader (Molecular Devices, Sunnyvale, USA) at 490nm. Inhibition rate (%) = [(A Control –A Treated)/ AControl] ×100%.
Annexin V-FITC/PI dual staining
HepG2 cells were treated with different concentrations of JS-K for 6 h, 12 h, and 24 h. Thereafter, cold PBS was used to wash the cells twice that subsequently were resuspended in 500 μL binding buffers. Annexin-V-FITC and PI were added to binding buffers for 10 min in the dark. The stained cells were analyzed by flow cytometry and analyzed by BD CellQuest Pro software (version 2.0, BD Pharmingen, BD Biosciences).
HepG2 cells were grown on a 48-well plate and resuspended with PBS in the absence or presence of JS-K. Then the cells were fixed with 4% paraformaldehyde and permeabilized with cold methanol for 15 min. Next, cells were aspirate methanol and rinsed in PBS for 5 min each, followed by incubating cells in 5% normal goat serum for 60 min. Subsequently, cells were incubated with the primary antibody at 4°C overnight. Then the cells were washed three times with PBS and incubated with Alexa Fluor 594-conjugated anti-rabbit IgG antibody for 2 h at room temperature in a humidified chamber. Cells nuclei were stained with DAPI and visualized by a fluorescence microscope.
Western blot analysis
HepG2 cells were washed with PBS and lysed for 30 min. Then cells were collected and centrifuged at 12,000 g for 40 min. The proteins of total cell lysis, nuclear proteins were obtained, respectively. Protein was separated by a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for 2 h and then transferred to a polyvinylidene difluoride membrane (PVDF, Millipore, USA) for 2 h. 5% non-fat milk in TBST (Tris-buffered saline and 1% Tween 20) was used to block the membranes for 2 h. After blocking, cells were cultured with primary antibodies (diluted at 1:1000) at 4°C overnight. HRP-conjugated goat anti-mouse IgG or goat anti-rabbit IgG were cultured for 2 h at a dilution of 1:10000. The proteins were visualized with ECL assay through a Bio-Rad gel imaging and analysis system.
RNA and DNA interference assay
RNA interference Cells were collected and re-suspended in a DMEM medium. A single-cell suspension was subsequently seeded in 12-well plates (2×105 cells/well). Cells were infected or transfected with CIP2A or PP2A in the serum-free medium the next day. Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was used to transfect cells (2×105 cells/well) with CIP2A or PP2A-C siRNA for 48 h before treatment with JS-K for 24 h. Then the cells were collected to determine the expression of proteins.
DNA interference Cells were infected or transfected with CIP2A or PP2A in the serum-free medium the next day. For transfection, 2.5μg of DNA was used for each transfection using lipofectamine 3000 reagent for 4 h. Then the infection medium was removed and replaced with a complete medium for 48 h. Next, cells were treated with JS-K for 24 h to observe the expressions of proteins by Western blot analysis. Negative control plasmid was treated according to the above methods.
Quantitative data were expressed as mean±SD and analyzed by one-way ANOVA. Multiple comparisons between the groups were performed using Tukey’s test. Statistical significance was set at a level of *P< 0.05 or **P < 0.01 level.