Cell line of HepG2
The human hepatoma cell line HepG2 was obtained from ATCC (American Type Culture Collection), and cultured with DMEM medium (Gibco, Carlsbad, CA, USA) and 10% FBS (Gibco, Carlsbad, CA, USA) in a humidified incubator with 95% air, 5% CO2− humidified atmosphere at 37℃. In experiment group, cells were treated with different concentration of RA. RA was purchased from Aladdin (R109804; China).
Western Blot Analysis
The total protein from cells was extracted using RIPA lysis buffer, then concentrations of total proteins were quantified with bicinchoninic acid assay. Total protein (50 µg) was added into 12% SDS-PAGE gel, further transferred onto polyvinylidene difluoride membranes. 5% non-fat dry milk in Tween-20 (0.1%)-containing TBS was used to block the membranes, then which was incubated with primary antibodies, further incubated using the HRP (horseradish peroxidase)-conjugated second antibodies (1:1000; Cell Signaling Technology, Beverly, MA, USA). Blots were visualized by electrochemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA, USA).
The following primary antibodies were used: Fyn (ab125016; Abcam, USA), PI3K (4249S; Cell Signaling Technology, USA), MMP-2 (87809S; Cell Signaling Technology), MMP-9 (13667S; Cell Signaling Technology), Bax (3498S; Cell Signaling Technology), Bcl-2 (2772S; Cell Signaling Technology), cleaved caspase-3 (9661S; Cell Signaling Technology), caspase-3 (9662S; Cell Signaling Technology), phosphorylated Akt (p-Akt) (Sc-514032; Santa Cruz, USA), Akt (Sc-81434; Santa Cruz), NF-κB p65 (Sc-8008; Santa Cruz), β-actin (Sc-47778; Santa Cruz), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Sc-47724; Santa Cruz).
Analyze viability of HepG2 cells
The viability of HepG2 cells was detected using CCK-8 method. Briefly, cultured primary cortical neurons were cultured in plates (5 × 103 cells/well). After 24 h of incubation, CCK-8 solutions were added into each well. After incubation for another 16 h, and then absorbance (450 nm) was measured with Microplate Reader.
Determine proliferation of HepG2 cells
The proliferation of HepG2 cells were detected with XTT (sodium 3'-[1-(phenylaminocarbonyl)-3, 4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) (Roche Applied Science, Mannheim, Germany). After cultured in 96-well plates (3 × 103 cells/well) for 48 h, HepG2 cells were dealt with plasmids or siRNA, then cell proliferation assay was performed, the absorbance (450 nm) was measured with microtiter plate reader (Bio-Rad)
BrdU assay and determine of mitotic entry
The transfection of siRNA or plasmids was conducted during the interval of two blocks with thyidine for avoid the its potential effect on cell cycle. The synthesis of DNA was determined with BrdU labeling, BrdU-positive HepG2 cells were manually scored with immunofluorescence microscope. Furthermore, according to DNA staining using time-lapse videomicroscopy, the events of mitotic entry were recorded through observing condensation of DNA and morphology of nucleus.
Determimation of apoptosis
Apoptotic rates of cells were determined with FITC Annexin-V Apoptosis Detection Kit I (BD, San Diego, California, USA). Briefly, cells were collected, washed with PBS and resuspended in binding buffer, double-stained with propidium iodide (PI) and Annexin V-FITC, followed by the apoptosis analysis using flow cytometry (BD Biosciences, San Jose, CA, USA).
Wound Healing Assay
Cell migration was evaluated by using the wound healing assay as described previously. Briefly, HepG2 cells were seeded in 24-well plates at 5 × 104 cells/well and allowed to adhere. Then, a wound was scratched by using a 200-µl pipette tip. After washing off the separated cells with phosphate-buffered solution (PBS), serum-free medium containing different concentrations of RA (0, 100, 200, and 400 µM) was added to the wells. The wound was observed at regular intervals between 0 and 48 hrs. Randomly selected areas were photographed by using a phase-contrast microscope (Olympus CKX41, Japan) and the wound area was calculated by ImageJ software (Bio-Rad, USA).
Invasion Assay
The invasion assay was conducted by using Corning® BioCoat™ Matrigel® Invasion Chambers (Catalog No. 354480; Corning, USA) with 8-µm pore chambers inserted into 24-well plates. HepG2 cells (5 × 104 cells) were cultured in 500 µl of serum-free DMEM with RA in the inserts and 500 µl of DMEM containing 15% FBS in the bottom of wells. After 24 hrs of incubation, the inserts were washed three times with PBS and the cells were fixed with 4% paraformaldehyde for 10 min. After washing with PBS, the cells were stained with 0.1% crystal violet for 15 min. After another wash with PBS, the inner sides of the chamber were wiped with a cotton swab and images of the cells that invaded through the Matrigel® were taken using a phase-contrast microscope. Finally, the number of invading cells was counted.
TUNEL and DAPI Staining
HepG2 cells were seeded on a cover slide in a 24-well plate at a density of 3 × 104 cells per well and kept in a CO2 incubator for overnight growth. Cells were treated with various concentrations of RA (0, 100, 200, and 400 µM) or vehicle for 24 hrs. Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining was performed according to the manufacturer’s instructions (C1086; Beyotime Biotechnology, China). Briefly, the cells were fixed with 4% paraformaldehyde for 30 min, incubated for 5 min with 0.3% Triton X-100 in PBS, and then incubated with a reaction mixture containing terminal deoxynucleotidyl transferase and fluorescent labeling solution for 60 min according to the manufacturer' s protocol. The cells were then stained with 4' ,6-diamidino-2- phenylindole (DAPI) for 5 min and mounted using ProLongTM Gold antifade reagent (P36930; Thermo Fisher Scientific, USA). Stained cells were analyzed using a confocal microscope (LSM 800; Zeiss, Germany)
Statistics
The continuous variables with normally distribute were indicated with mean ± SD (standard deviation). GraphPad Prism 5.0 was used to conduct statistical analysis. For comparisons between two groups, Student’s t-test was performed. Moreover, one-way analysis of variance (ANOVA) with Kruskal-Wallis post hoc tests were performed for multiple group comparisons to compare and analyze quantitative data. Besides, abnormally distributed data between two groups were analyzed with Kruskal-Wallis analysis of variance method. P < 0.05 was considered as difference with statistically significance.