Cell Culture and cell culture reagents
Two Human kidney cell lines, A498 (cat. no. HTB-44, ATCC, Inc, USA) and 786-O (cat. no. HTB-44, CRL-1932, Inc, USA) were maintained in DMEM (cat. no. 11965092, ThermoFisher, Inc, USA) supplemented with 10% FBS (fetal bovine serum, cat. no. 30067334, ThermoFisher, Inc, USA) and 2mM glutamine (cat. no. 25030164, ThermoFisher, Inc, USA). These cells were cultured in DMEM supplemented with 10% FBS, 2mM glutamine, 1% Pen/Strep (cat. no. 15070063, ThermoFisher, Inc, USA). All cells were grown in collagen-coated plastic (50ug/mL) at 37°C in a humidified 5% CO2 atmosphere.
Cell viability was detected by Cell Counting Kit-8 assay
CCK-8 assay kit (cat. no. C0038, Beyotime, Inc, China) was used to detect cell viability. According to the manufacturer's instructions, logarithmic growth phase A498 or 786-O cells were seeded in 96-well plate at a density of 2000 cells/well. Embelin (cat. no. 178493, Sigma, Inc, USA) or Axitinib (cat. no. PZ0193, Sigma, Inc, USA) of different concentrations were added, and 6 compound holes were set up for each concentration. The cells were cultured at 37 °C for 24h. Then cells were incubated with 100 µL of medium containing 10% CCK-8 reagent for 2h. The absorbance values of each well at 450 nm were detected through a spectrometer.
Cell proliferation was detected by MTT assay
MTT Cell Proliferation Assay Kit (cat. no. C0009S, Beyotime, Inc, China) was used to detect cell proliferation following manufacturer's instructions. Cells were seeded into 96-well plates at 2×103 per well and incubated under standard culture condition for 72h. Following Embelin or Embelin treatment, 10 μl of MTT solution was added to each well and the plates were incubated for another 3 h at 37°C, and formazan crystals were dissolved with 100 μl of 0.04 N HCl-isopropanol. After incubated for 4h at 37°C. The absorbance of individual wells was read at 570 nm test wavelength using a microplate reader (Victor Nivo 5s, Perkinelmer, Inc, Finland). Cell proliferation activities were determined by absorbance values.
Cell survival was tested by crystal violet assay
Crystal violet assay was used to detect Cell survival in different concentration of drugs.[48] Briefly, cells were seeded on 48 well cell culture plates at 5 × 103 cells/well and incubated at 37 °C for 24 h. After the addition of drugs, cells were incubated at 37 °C for 2 days. At the indicated time points, cells were fixed for 20 min with 4% paraformaldehyde (cat. no. P0099, Beyotime, Inc, China). Subsequently, wells were washed by submersion in a water bath. Then wells were aspirated and 0.5% crystal violet (cat. no. C0121, Beyotime, Inc, China) was added. After 20 min, crystal violet was aspirated and wells were thoroughly washed by submersion in a water bath. Plates were left to dry at 37°C and crystal violet was reconstituted in 10% acetic acid, image of each well was captured and analyzed using ImageJ software.
Apoptosis was assessed by Annexin V-FITC staining
Annexin V-FITC staining kit (cat. no. C1062S, Beyotime, Inc, China) was used to detect cell apoptosis. Following manufacturer's instructions, cells were treated with indicated concentrations of drugs for 48 hr. Then cells were collected, washed 3 times with PBS and re-suspended in PBS. Each sample was incubated with 5 μl of Annexin V-FITC and 10μl propidium iodide staining solution in 195 μl of the binding buffer for 15 min at room temperature in the dark. Then, the samples were analyzed by flow cytometry (ACCURI C6, BD Bioscences, Inc, USA).
Protein expression level was detected by western blot assay
Western blot assay was used to detect protein expression levels. Proteins were extracted using RIPA total protein lysate (cat. no. P0013C, Beyotime, Inc, China) following the manufacturer's instructions. Proteins from each sample were separated by electrophoresis on 10% SDS-PAGE gels and transferred onto PVDF membranes (cat. no. FFP39, Beyotime, Inc, China). After incubation with blocking solution (cat. no. P0252, Beyotime, Inc, China), the PVDF membranes were separately incubated with anti-BCL-2, anti-BAX, anti-cleaved caspase-3, anti-GAPDH and anti-HIF-1α (cat.no. ab32124, ab32503, ab32042, ab9485, ab51608, Abcam, Inc, USA,1:2000) at 4°C overnight. After the blots were washed, they were incubated with HRP-conjugated Goat Anti-Rabbit IgG H&L secondary antibody (cat.no. ab6721, Abcam, Inc, USA,1:2000) for 1.5 h and detected by ECL like Western reagent (cat. no. P0018S, Beyotime, Inc, China) with a chemiluminescent imaging system (ChemiDoc MP, Bio-Rad, Inc, USA).
The target pathway of Embelin was predicted by network pharmacology analysis
To predict the signaling pathways that embelin acts on, three networked pharmacological databases were used for analysis. Embelin was retrieved from CTD Database (Comparative Toxicgenomics Database, http://ctdbase.org/), STITCH Database (http://stitch.embl.de/) and BATMAN-TCM Database (Bioinformatics Analysis Tool for Molecular mechanism of Traditional Chinese Medicine, http://bionet.ncpsb.org.cn/batman-tcm/) to obtain the corresponding signaling pathways. Subsequently, "Kidney Cancer" was retrieved from the CTD Database and the corresponding signal pathway was obtained. Winn diagram was used to analyze the results of four retrievals and the intersection was selected as the most likely target pathway of embelin.
Statistical analysis
The GraphPad Prism software (version 8.0) was used to perform Statistical analysis and mapping. One-way analysis of variance followed by a Bonferroni post-hoc test was used to analyze differences between groups. Data were considered significantly different when p < 0.05. Image analysis was conducted using ImageJ software (1.53c).