1.Cell culture
HCT116 cells (Hunan Fenghui Biotechnology Co., Ltd, Hunan, China) were cultured in Dulbecco's Modified Eagle's Medium (DMEM,Gibco, USA) containing 10% fetal bovine serum (FBS༌Gibco, USA) and 1%penicillin/streptomycin (Gbico, USA) in a 37 °C, 5% CO2 incubator environment.
2.Cell viability assay
MTT assay was used for cell viability assessment. HCT116 cells seeded in 96-well plates at 5000 cells/well. After incubation 24 h treatment with various concentrations of Aloe-emodin(AE was provided by Lot.Y02M11Y16995,Shanghai Yuanye Biological Technology Co., Ltd. Shanghai, China. HPLC ≥ 97%)at 24 h, 48 h, or 72 h and 0.5 mg/ml MTT༈Sigma༌USA༉ at 37℃ under 5% CO2 for 4 h and then add DMSO 100 ul ༈Guangdong Guanghua Technology Co., Ltd., China༉to each well. Immediately measure its absorbance at 490 nm and calculate its cell viability, the percentage of cell viability according to the following formula, OD value of the treated cells/ OD value of the control cell × 100%. By definition, the viability of the control cells from the untreated cultures was defined as 100%.
3.Colony formation
Measure the effect of cell proliferation through colony formation experiments. The HCT116 was evenly spread in a 60 mm culture dish with 1 × 105 and cultured in complete medium for 24 h, treated with various concentrations of aloe-emodin for 24 h, 48 h, and cultured for 7 days. The cells were stained with crystal violet (Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China) for 15 min and photographed.
4.DAPI staining
DAPI staining can be used to observe apoptosis, penetrate the cell membrane and double-stranded DNA in the nucleus to play a role in labeling. First, HCT116 cells were seeded on 6-well plates at 1 × 105 per well. After 24 hours of culture, the cell was treated with various concentrations of aloe-emodin for 24 h, 48 h. The morphological changes of the cells were observed under a microscope, then fixed with ethanol(Hangzhou Chemical Reagent Company, Hangzhou, China) and PBS (Hangzhou Northrend Biotechnology Co., Ltd., Hangzhou, China,) washed, cells were stained with DAPI (10 mg/mL) (4′,6-diamidino-2-phenylindole, Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China) for 20 min in the dark, and then photographed by fluorescence microscope(EVOS FL,USA).
5.Caspase-3 Activity Assay
Caspase-3 is a key enzyme in the process of cell apoptosis. Use the caspase-3 activity kit (Nanjing Jiancheng Biotechnology Co., Ltd., Nanjing, China). for the cultured HCT116 and determine the caspase-3 activity in the Cell lysate according to the protocol provided by the manufacturer. The principle is to change the sequence-specific polymorphism of caspase-3 (Ac-DEVD-pNA) is coupled to the yellow group pNA. When the substrate is cleaved by caspase-3, the yellow group pNA is released, and then the caspase-3 activity is measured by the absorbance value of 405 nm in the microplate reader (Synergy H1, Biotek, USA).
6.Cell cycle by flow cytometry
The distribution of cells in the cell cycle is assessed by flow cytometry. HCT116 was spread on a 6-well plate and cultured overnight. It was treated HCT116 cells with different concentrations of Aloe-Emodin for 24 h and 48 h. The adherent cells were digested with trypsin containing EDTA (Gibco, USA), collected in complete DMEM medium (Gibco, USA), centrifuged, washed with PBS (Hangzhou Northrend Biotechnology Co., Ltd., Hangzhou, China), fixed with ethanol at -20 °C overnight, treated with PI/Rnase (BD Biosciences, San Diego, USA), and analyzed by Beckman flow cytometer (Beckman, USA) cell cycle.
7.Western blotting
HCT116 cells were seeded in a 6-well plate, treated with different concentrations of aloe-emodin for 24 hours, washed with pre-chilled PBS, and then lysed in lysis Solution (Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China) on ice for 30 minutes then collected the cells. The protein concentration was established by the BCA method. The proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to the PVDF membrane. The membrane was blocked with 5% skimmed milk powder (Shanghai Yuanye Biological Technology Co., Ltd. Shanghai, China.)and then with the primary antibody overnight at 4 °C.Then the membrane was washed in TBST and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 2 h at 37℃. Finally, the immunoreactive band by using ECL (Affinity Biosciences, Changzhou, China)exposed on the Chemiluminescence digital imaging system (Bio-Rad, USA).Then use Image J 1.51 k software to qualitatively and quantitatively analyze the protein bands.
8.Statistical analysis
All data were presented as mean ± SD, and statistical analysis was carried out by GraphPad Prism 8 software. The one-way ANOVA analysis was implemented to demonstrate differences between groups (P < 0.05).