Chemical compounds and reagents
Emodin was purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China; Purity ≥ 98% by HPLC; lot number: T17O11F127680), which was dissolved in DMSO to provide a stock 50 mM solution and stored at -20°C. Fetal bovine serum, culture medium, and other solutions used for cell culture, where specially noted, were from Gibco (Brazil). Chloroquine (CQ), an autophagy inhibitor, was purchased from Selleck Chemicals (TX, USA). Matrigel was purchased from Corning (NY, USA). Dimethyl-sulfoxide was obtained from Sigma-Aldrich Chemical (MO, USA). EdU detection kit and ad-mCherry-GFP-LC3 were purchased from RiboBio Co., Ltd. (Guangzhou, China). Annexin V/PI kit and cell cycle detection kit were purchased from BestBio (Shanghai, China). Trizol was obtained from Invitrogen (CA, USA). The primary antibodies against E-cadherin, N-cadherin, vimentin, PI3K, p-PI3K, AKT and p-AKT were bought from Abcam (Cambridge, MA, USA). The primary antibodies against β-catenin, GSK3β, p-GSK3β, Snail, Slug, Beclin1, LC3B, SQSTM1, and p-mTOR were purchased from Cell Signal Biotechnology (Beverly, MA, USA). HRP-conjugated Affinipure Goat Anti-Mouse IgG and Goat Anti-Rabbit IgG were sourced from Proteintech (Chicago, IL, USA).
Cell line and cell culture
The human liver cancer cell lines HepG2 and Bel-7404 were obtained from the Chinese Type Culture Collection (CTCC, Shanghai, China). The cells were cultured in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The Human normal liver cell line L02 (CTCC, Shanghai, China) was maintained in 10% FBS-supplemented DMEM medium and 1% Penicillin/Streptomycin. All the cell lines were incubated under standard conditions at 37°C in a humidified atmosphere of 5% CO2. The cells were passaged twice or thrice a week and discarded after 20 passages. All cells have been tested negative for mycoplasma contamination using Mycoplasma Detection Kit (Beyotime Biotechnology, Guangzhou, China).
Cell viability assay
Cells were digested and inoculated in 96-well plate at a density of 6000 cells/well, incubated overnight. Afterward, emodin at different concentrations (0, 25, 50, 75, and 100 µM) were used to treat cells for 24, 48, and 72 h. Finally, the cell viability was determined by MTT (Beyotime Biotechnology, Guangzhou, China). Each experiment was repeated three times, and for each experiment 6 replicates were prepared. IC50 is defined as the sample concentration at which 50% inhibitions were reached.
Cell proliferation assay
EdU cell proliferation staining was performed using an EdU kit. Briefly, HepG2 cells were seeded in 24-well plates at 1 × 105 cells/mL and treated with four different concentrations of emodin chosen below its 48h IC50 value, i.e., 0, 15, 30, 60 µM for 48 h. Subsequently, cells were incubated with EdU for 2 h, fixed with 4% paraformaldehyde for 15 min, and permeated with 0.3% Triton X-100 for another 15 min. The cells were incubated with the Click Reaction Mixture for 30 min at room temperature in a dark place and then incubated with Hoechst 33342 for 10 min.
Cell apoptosis assay
Cell apoptosis staining was performed using the cell apoptosis detection kit according to the manufacturer’s instructions. The cells were washed twice with phosphate-buffered saline (PBS). 1X Binding buffer was added to 1 × 106 cells/mL, and PI was then added in the dark. After incubation for 30 min in the dark, the cells were immediately analyzed using CytoFLEX flow cytometer.
Cell cycle analysis
At the end of incubation, cells were trypsinized. Cells were collected by centrifugation at 1000 g for 5 min, washed twice with ice-cold PBS, and then fixed with 70% cold ethanol and stored at 4°C for 24 h. Cells were centrifuged again, washed with cold PBS twice, incubated with RNase A (0.1 mg/mL) for 1 h at 37°C, and stained with PI (0.1 mg/mL) for 30 min in the dark. The DNA content was measured by flow cytometry, and the percentage of cells in each phase of the cell cycle was evaluated using CytoFLEX flow cytometer and ModFit software.
Colony formation assay
A density of 400 cells per well seed onto 6-well culture plate and emodin in DMEM medium free of FBS was added to the culture at 3 days after seeding. The culture was continuously maintained for another 14 days and subjected to the colony formation assay. Plates were imaged and colonies were enumerated using Image J (National Institute of Health, USA).
Wound healing assay
HepG2 cells were seeded in 6-well plates at the density of 3 × 105 cells per well, grown to reach 95% confluency monolayer, and then a “scratch” was made in the cell monolayer using a 100 µL pipette tip to create a wound before drug treatment, afterward the detached cells were washed out using PBS. All wells were treated with different concentrations of emodin. In the same field, the wound distance was measured at 24 h, and 36 h, under DMi1 inverted microscope at 5X magnification (Leica, Germany) to evaluate wound closure. Wound healing was analyzed using Image J. Experiments were carried out in triplicate and repeated at least three times.
Transwell Migration and Invasion Assays
For the Transwell invasion assay, serum-free medium was applied to dilute the Matrigel (1: 9), and then 50 µl of diluted Matrigel was inoculated into each chamber. After being treated with or without emodin, HepG2 cells were digested and resuspended at a density of 2.5 × 105 cells/mL with DMEM medium without FBS. Then, we added 0.2 mL of the cell suspension to each upper chamber of the 24-well plate, while 0.6 mL DMEM containing 20% FBS was added to the lower chambers. After 24 h, the upper chambers were washed, fixed with 4% paraformaldehyde for 20 min, stained with 0.25% crystal violet for 30 min, and imaged by a microscope. Four fields (10X) were selected randomly, and the number of HepG2 cells was counted. For detection of the migrating ability of cancer cells, the protocol was the same as that for the invasion but without Matrigel.
Western blot analysis
HepG2 cells were seeded at 1 × 106 cells per T25 flask and incubated overnight. After 48 h of treatment with concentrations of emodin, cells were washed with PBS, and digested with trypsin for 20 min at 4 ℃. Cell lysates were centrifuged (12,000 rpm) for 30 min at 4 ℃. The protein samples were assessed with BCA Kit, and protein lysates were boiled at 95 ℃ for 10 min and separated on SDS-PAGE. Then, they were electro-transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in 5% milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at room temperature and washed in TBST for three times. The blots were incubated with primary antibodies overnight at 4 ºC. The primary antibodies against E-cadherin, N-cadherin, Vimentin, Snail, Slug, β-catenin, Beclin1, LC3B, SQSTM1, p-AKT, AKT, p-PI3K, PI3K, p-GSK3β, GSK3β, p-mTOR, and β-actin at appropriate dilutions as recommended by the manufacturer. After the membranes were washed in TBST three times, the blots were then incubated with HRP-conjugated Affinipure Goat Anti-Mouse IgG or Goat Anti-Rabbit at room temperature for 1h. Proteins were visualized by chemiluminescence technology and analyzed with the Image J.
HepG2 cells were seeded on 35 mm diameter confocal dishes until they reached about 70% confluence and then transfected with ad-mCherry-GFP-LC3B adenovirus at an MOI of 20 for 6 h at 37°C. Following treatment with emodin (60 µM) for 48 h, Finally, images were taken with a confocal microscope (Leica, Germany).
HepG2 cells in 6 different petri dishes were divided into two equal groups. After being treated with emodin for 48 h, total RNA was extracted from treated and untreated cells using Trizol reagent. Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit) was used to do the total RNA sample QC. Then we assembled those clean reads into Unigenes, followed with Unigene functional annotation, SSR detection and calculate the Unigene expression levels and SNPs of each sample. Finally, we identify differential expressed genes (DEGs) between samples and do clustering analysis and functional annotations. DEGs at each stage or site were used for further analyses of gene ontology (GO) and KEGG pathways using the Database for Annotation, Visualization, and Integrated Discovery (DAVID: http://david.abcc.ncifcrf.gov).
Each experiment was performed at least three times, and all data are presented as the mean ± SD. Statistical analysis was done using GraphPad Prism Version 8.1.0 (CA, USA). One-Way ANOVA tests were used to assess the differences between the means of treated and untreated cells or groups. P-values < 0.05 were considered significant (*, P < 0.05; **, P < 0.010; ***, P < 0.001).