2.1 Cell culture and Reagents
Human RCC cells (ACHN and A498) and the normal renal cell line (HK-2) were obtained from Guangzhou Jennio Biological Technology Co., Ltd. (Guangzhou, China). ACHN and HK-2 cells were grown in Dulbecco’s modified Eagle medium (DMEM) (GIBCO, Thermo Fisher Scientific, Inc., Waltham, MA, USA). A498 cells were cultured in RPMI 1640 medium (GIBCO, Thermo Fisher Scientific, Inc., Waltham, MA, USA). All culture media were supplemented with 10% (v/v) fetal bovine serum (FBS; GIBCO, Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37 °C in a humidified atmosphere that contained 5% CO2. Berberine was purchased from Sigma (St. Louis, USA). DMSO was used to dissolve berberine to stock solutions with a concentration of 100 mM. N-acetylcysteine (NAC) was purchased from the Beyotime Institute of Biotechnology (Shanghai, China). PBS was used to dissolve them to 100 mM respectively. These stock solutions were stored at -20 °C for experiment use.
2.2 Cell viability assay
Methyl-tetrazolium (MTT) assay was used to detect the viability of RCC cells. Cells were seeded in 96-well plates (4 × 103 cell/well) (Nest Biotechnology, Wuxi, China), next day, various concentrations(0, 10, 20, 50, 100, 200 µM) of berberine were used to incubate these cells for 24 h, 48 h, and 72h, then, add 20 μL MTT (Sigma Aldrich, St. Louis, MO, USA) in every which need to test. After 4 hours, a multiscan Ascent microplate photometer (EnSpire 2300 Multilabel Reader, PE, USA) was used to measure the absorbance of the solution at 492 nm.
2.3 Colony formation assay
After being treated with various concentrations (0, 20, 50, 100 µM) of berberine for 24 h, ACHN and A498 cells were trypsinized and 2 × 103 cells were plated into 6-well plates (Nest Biotechnology) for another 2 weeks (ACHN) and 10 days (A498). The cells were stained with crystal violet (Beyotime, Shanghai, China) for 20 min after being fixed with 4% paraformaldehyde for 15 min. Finally, the number of colonies formed was counted.
2.4 Cytotoxicity assay
ACHN cells(4 × 103 cells/well) and A498 cells (4 × 103 cells/well) were seeded in 96-well plates and treated with different BBR concentrations (0, 20, 50, 100 µM) for 24 h. After centrifuging at 400 × g for 5 min, the supernatants (120 μL/well) were transferred into new 96-well plates, and 60 μL of lactate dehydrogenase(LDH) detection reagent was added to each well. The plates were incubated for 30 min at room temperature in the dark, and the absorbance of the formazan was detected at 490 nm using a reader (EnSpire 2300 Multilabel Reader, PE, USA).
2.5 Caspase-Glo 3/7 assays
ACHN cells(4 × 103 cells/well) and A498 cells (4 × 104 cells/well) were seeded in 96-well plates and treated with BBR(0, 20, 50, 100 µM) and N-Acetyl-L-cysteine (NAC) (100 µM) for 24 h. Equal volumes (100 µL) medium and caspase-Glo 3/7 reagent (Promega Corporation, Madison, WI, USA) were added to each well, and the cells were incubated at room temperature in the dark for 30 min. Luminescence was measured by a luminometer (Berthold Sirius L; Titertek-Berthold, Pforzheim, Germany).
2.6 ATP levels
The adenosine triphosphate (ATP) levels were detected by the ATP Assay kit(Beyotime Institute of Biotechnology) in ACHN and A498 cells. Cells (1 × 105 cells/well) were treated with different BBR concentrations (0, 20, 50, 100 µM) for 12 h. Subsequently, cells were treated with 200 µL lysis buffer (Beyotime Institute of Biotechnology) and collected by centrifugation at 12,000 × g for 5 min at 4 °C. Subsequently, 50 µL supernatant and 100 µL ATP detection reagent were mixed. Firefly luciferase activity was measured using a luminometer (Berthold Sirius L).
2.7 Apoptosis assay
The apoptotic rate of ACHN and A498 treated with various concentrations(0, 20, 50, 100 µM) of berberine for 24 h were detected using the fluorescein isothiocyanate (FITC)-labeled Annexin V Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, cells were digested with Trypsin Solution without EDTA (Beyotime), then, washed and centrifugated according to the manufacturer’s instructions. FITC-labeled annexin V and propidium iodide were used to stain cells for 15 min. The apoptotic death was determined by annexin V/ PI staining. Then the apoptotic rate of cells was measured by Flow Cytometer (BD FACSCelesta, Becton Dickinson USA) using flow cytometry BD FACSDiva 6.1 software.
2.8 Wound healing assay
Cells were seeded into 6-well plates and scratches were made with a pipette tip after the confluence of cells up to 100%. Then, washed cells with PBS, a medium without FBS were used to culture cells. After being treated with various concentrations(0, 20, 50, 100 µM) of berberine for 24 h, the wound gaps and cells were scanned under EVOS XL Core Imaging System (Life Technologies, USA). The areas of wounds were assessed by ImageJ software. The wound healing rates were measured by ( ([original wound area of drug group]-[wound area of drug group])/ ([original wound area of control group]-[wound area of control group]) )× 100%.
2.9 Transwell assay
ACHN and A498 were harvested after being treated with various concentrations(0, 20, 50, 100 µM) of berberine for 24 h. Then, the cells were resuspended with serum-free medium and then 200 μL cells suspension(2 × 104 cells) were plated in the upper chamber of the insert (membrane pore size, 8 μm; Corning) with Matrigel (BD Biosciences, Billerica, MA, USA). 750 μL medium with 20% FBS was added to the bottom chambers. After incubating for 24 hours at 37°C, the cells were fixed with 4% paraformaldehyde and stained with crystal violet. Cells adhering to the base membrane of the inserts were scanned in 5 random fields with EVOS XL Core Imaging System. The migration cell numbers were then counted with ImageJ software.
2.10 Intracellular ROS detection
The level of ROS in cells was explored using the Reactive Oxygen Species Assay Kit (Beyotime). Cells were gathered and suspended with a medium (without FBS) that contained DCFH-DA (10 µM) after being treated with various concentrations(0, 20, 50, 100 µM) of berberine. Then, cells were incubated at 37 °C for 20 min in dark and shaken every 3-5 min. Serum-free mediums were used to wash cells three times to clear intracellular DCFH-DA. The intensity of intracellular ROS was determined by the Fluorescence intensity of DCF which was measured by Flow Cytometer (BD FACSCelesta, Becton Dickinson USA) using flow cytometry BD FACSDiva 6.1 software.
2.11 Western blot analysis
Proteins were extracted from ACHN and A498 treated with various concentrations of berberine for 24 h. In brief, cells were lysed in the mixture of RIPA Lysis Buffer (Beyotime) and PMSF ((phenylmethanesulfonyl fluoride) (Beyotime), and the concentrations of proteins were detected using Bradford Protein Assay Kit (Beyotime, Shanghai, China). Then, the proteins were separated by SDS‐PAGE (SDS-poly-acrylamide gel electrophoresis) and transferred onto PVDF (polyvinylidene fluoride membranes) (EMD Millipore, Billerica, MA, USA). Next, 5% non-fat milk in TBST (Tris-buffered saline and Tween 20) was used to block the membranes for 1 h, and the membranes were blotted with primary antibodies at 4 °C overnight. Primary antibodies (dilution 1: 1,000) for Caspase-3 (#9665S), Caspase-9 (#9502S), Bad (#9239), Bax (#2772S), Bak (#12105S), Cyto-c(#4272S), Bcl-2 (#2876S), TIMP-1 (#8946S), E-cadherin (#3195S), Vimentin (#5741S), N-cadherin (#4061S), Snail (#3879S), γH2AX (#2577S), Rad51 (#8875S), PCNA (#2586S) (all Cell Signaling Technology, Inc., Danvers, MA, USA) were diluted with Primary Antibody Dilution Buffer (Beyotime). IgG-HRP secondary antibody (EarthOx, USA) was used to incubate the membranes for 1 h at room temperature. Proteins were observed in Tanon 5200 chemiluminescent imaging system (Shanghai, China).
2.12 Immunofluorescence analysis
Cells were treated with various concentrations(0, 20, 50, 100 µM) of berberine for 48 h, then washed with PBS and fixed with 4% paraformaldehyde, Triton X-100 was used to incubate cells for 30 min, next, the cells were treated with PBS containing 1% BSA (Solarbio, Beijing, China) for 1 h. Cells were incubated with 1% BSA containing anti-γH2AX polyclonal Ab (dilution 1:100) (CST) at 4 °C overnight. In addition, Alexa Fluor 488-conjugated anti-rabbit IgG (dilution 1:100 ) (Thermo Fischer Scientific, Carlsbad, CA, USA) was used to treat the cells for 2 h at room temperature in dark. Then, cells were washed with PBS three times and stained with the Mounting Medium, antifading (including DAPI) for 3 min. Finally, cells were observed using a confocal laser scanning microscope. (Olympus FV3000, Tokyo, Japan).
2.13 Statistical analysis
The analysis of all data was performed with SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA). A one-way ANOVA test was used to determine the significance. *p<0.05, **p<0.01, and ***p<0.001 were considered statistically significant. Data were shown as the means ± standard deviation. All experiments were performed in triplicate.