2.1 Cell culture and reagents

Colonic carcinoma cell line HCT-116 was purchased from Shanghai Genechem Co., LTD. MCCOY'S 5A medium was used for culturing the cell line. 10% fetal bovine serum and 100 U/mL penicillin–streptomycin were added to supplement the medium.

We purchased RIPA buffer, GRg3, MCCOY'S 5A medium, and MTT Kit from Beijing Solarbio Science & Technology Co., Ltd. FBS was purchased from Biological Industries Israel Beit Haemek Ltd. Enhanced BCA protein assay kit, SDS-PAGE Electrophoresis Buffer, TBST, Precast PAGE Gel and the following antibodies: JAK3 Rabbit Polyclonal Antibody (AF7314), Phospho-Stat5 (Tyr694) Rabbit Monoclonal Antibody (AF2005), STAT5a Rabbit Monoclonal Antibody (AF2038), STAT5b Rabbit Monoclonal Antibody (AF2647), Goat Anti-Rabbit IgG H&L(HRP) (A0208) were bought from Beyotime Biotechnology lnc (Shanghai, China). GAPDH antibody and Goat Anti-Mouse IgG Antibody, H&L-HRP conjugate were bought from Proteintech (Wuhan, Hubei, China).

2.2 MTT assay

A colorimetric MTT [3-(4,5-Di-2-yl)-2,5-tetrazolium bromide] test was carried out to evaluate the efficacy of several concentrations of GRg3 on the viability of HCT-116 cells.

Briefly HCT-116 cells ( 5 × 103 cells/well) in the logarithmic growth phase were added to ELIZA 96 well plates and Several concentrations of GRg3 (10, 20, 30, 40, 50, 60, 70, 80 μmol/L) were added for 48 h. After ending incubation time, consequently, 10 μl MTT (solution was prepared in sterile PBS), was added to the plates and incubated for 4 h. The reaction was stopped by adding 100 μl DMSO and OD (optical density) values were read at nm with an ELISA reader at 490nm.

2.3 Colony formation assay for carcinoma cells

For this test, HCT-116 cells were trypsinized and harvested, then (1,000 cells/well) seeded in 6-well plates. We used a drug-free medium and Ginsenoside RG3 (50 μM)for approximately 2 weeks of incubation. Three replicates were set for each group of cells. Formalin was added to fix the colonies in 30´ and colonies then were stained with Giemsa for 30´. Colonies were photographed and counted under the microscope and those containing > 50 cells were considered effective.

2.4 Flow cytometry assay

The inhibitory efficacy of RG3 on the cell cycle was analyzed with propidium iodide (PI) uptake and flow cytometry.

The HCT-116 cells were exposed to 50μM GRG3 for 48 h. Then cells were washed with cold PBS and fixed with cold 70% ethanol for 24 h. Propidium iodide (PI) and RNase A for 30´ at 37°C were used to stain the cells. Flow cytometry assay was used to detect DNA content.

2.5 Trans well cell migration assay

The migration inhibitory efficacy of GRg3 on the HCT-116 cell line was determined with a trans well assay. For this assay, serum-free medium was used to re-suspend the digested cells, and about 5 × 104 cells were inoculated in each trans well chamber. The serum concentrations in the upper and lower chambers were 0.9% and 10%, respectively, and the cells were exposed to GRg3 (60μM) for 24 h. Then fix cells for 30´ in 10% formalin, dye with 0.05% crystal purple solution for 20´, clean it with a wet cotton-tipped applicator, and remove non-migrated cells gently from the apical side of the membrane. Repeated this process to make sure to eliminate all non-migrated cells. Then air dried and took pictures. The dye was dissolved with 70% ethanol and added to each well in a 96-well plate, and after 10–15 min the OD value of each well was read at 570nm of the enzyme labeling instrument to obtain the accurate value.

2.6 Cell scratch wound assay

Cell migration capacity was calculated by Cell scratch wound assay. Briefly, 9 × 105 cells/well of HCT-116 cells in their growth logarithmic phase were added to a 6-well plate. When cells were adhered to the wall, a straight line was drawn on the cells with the gun head of 1ml and washed with PBS solution 3 times. The relative distances of migration of cells were computed by taking pictures under the microscope after being cultured with GRG3 (50 μM) for 0 and 24 h. Experiments were performed in triplicate to reduce the errors.

2.7 Transcriptome sequencing and analysis of differentially expressed genes

HCT-116 cells were cultured for 36 h with a concentration of 50 μmol / L RG3 when the cells covered 40% of the bottom of the culture bottle. After washing with PBS solution, the total RNA of the cells was extracted with 1ml Trizol and set three repeats. The transcriptome sequence procedure was carried out by Shanghai Majorbio Bio-pharm Technology Co., Ltd. After the quality identification of RNA, the database was established, and the transcriptome was sequenced by the Illumina platform. The source of the reference gene was Homo sapiens (GRCh38). The differentially expressed genes were analyzed with DESeq2 software and the threshold for screening of | log2FC |≥ 0.585 and P < 0.05 was considered statistically significant.

2.8 Prediction of target genes of GRg3

The SMILES structure and 3D structure of GRG3 were searched by PubChem database (https://pubchem.ncbi.nlm.nih.gov), and direct action targets of drugs were obtained by using the SWISS Target Prediction (http://swisstargetprediction.ch/), STITCH (http://stitch.embl.de/) and the Pharmmapper databases (http://www.lilab-ecust.cn/pharmmapper/). In addition, the indirect targets were obtained by PPI of direct targets one by one (interaction score > 0.90) through the STRING database (https://string-db.org/), and all of the GRG3 target genes were obtained.

2.9 Key target acquisition and biological pathway analysis

The differential genes obtained by transcriptome sequencing and the target genes of GRG3 predicted by network pharmacology were intersected by Venny2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/index.html) to obtain the key targets of GRG3 that affect the HCT-116 cells reproduction and immigration. The involved biological pathways in the key targets were analyzed by the KEGG Mapper website. Protein–protein interaction (PPI) was constructed from the differential expressed genes and the target genes using the STRING database. Hub genes were analyzed and the interaction network was visualized by Cytoscape and CytoHubba.

2.10 Western blot assay

The cell line HCT-116 was exposed to 50 umol/L GRg3/vehicle for 36 h. The whole proteins were extracted from the cell by RIPA buffer containing protease inhibitor cocktail. An enhanced BCA protein test kit [Beyotime (Shanghai, China)] was used to determine the protein concentration. Precast PAGE Gel (Shanghai, China, Cat No. P04655) with a voltage of 130V for 90´ was used to separate the protein. GAPDH was used as the internal reference. It was transferred to a PVDF membrane with a current of 200 mA for 1 h. The membrane was blocked in TBST solution with skimmed milk (5%) for 2 h. Then they were overnight incubated with primary antibodies at 4°C. We washed the membrane with TBST three times and about 10 min per time. Secondary antibodies were added at room temperature. Two hours later, we used TBST to wash the membrane and dripped the ECL developer on the PVDF membrane evenly. Then development was performed using a gel imager.

2.11 Statistic analysis

Statistical analyses were done with Graph Pad Prism version 6.01 Software. Data were expressed by mean ± standard deviation (‾x ± s) and unpaired Student's t-test was used to analyze the differences and a P value < 0.05 was considered statistically significant. Deseq2 software was used to analyze the differentially expressed genes. The cut-off criteria were: | log2FC |≥ 0.585 and P < 0.05.