Preparation of RAC extract

RAC was purchased from Hangzhou Huadong Traditional Chinese Medicine Decoction Pieces Co., Ltd (catalogue number: 190211). 1000 g RAC was weighed, dissolved in 5000 mL of ethanol, refluxed for 10 h, extracted 3 times, and the extracts were combined, then evaporated, dried, gradient elution, and the solvent was removed, and the powder was collected and vacuum-dried for 24 h. 100 mL of distilled water was added to 100 g of dry powder, and then the stock solution was diluted to 3.16 mg/mL, 5.62 mg/mL, 10 mg/mL, 15.84 mg/mL, 31.6 mg/mL, 56.23 mg/mL, 100 mg/mL, followed by sterile filtration with a 0.22 μm microporous membrane, divided into packages, and stored in a 4 °C refrigerator for later use.

Cell culture

The 786-O cell lines (iCell-h235, Homo sapiens, CVCL number: CVCL-1051) and A498 cell lines (iCell-h235, Homo sapiens, CVCL number: CVCL-1056) were obtained and performed a Short Tandem Repeat (STR) profiling from iCell Bioscience Inc (Shanghai, China). 786-O and A498 cells were cultured in RPMI 1640 medium (FI201-01, TransGen Biotech, China) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin–streptomycin at 37 °C and 5% CO2. When the cell growth density reached 80%, the old medium was discarded, digested with 0.25% trypsin, and resuspended by adding fresh medium.

Cell counting kit-8 (CCK-8) assay

The logarithmic phase cell suspension was inoculated into 96-well plates and cultured for 24 h, then grouped into different concentration RAC groups: 0, 3.16, 5.62, 10, 15.84, 31.6, 56.23, 100 mg/mL RAC groups or 0, 1, 5, 10, 15, 30, 50 mg/mL RAC groups. Cells were also divided into different culture time groups: 0, 4, 8, 12, 24, 48, 72 h groups. After dosing with different concentration of RAC or culturing for a corresponding time, 10 μL CCK-8 solution (HY-K0301, MCE, USA) was added and incubated for 2 h. The absorbance at 450 nm was measured, and the cell viability was calculated. Five replicate wells were assayed in parallel.

Colony assay

The cells in the logarithmic phase were digested, seeded at 500–1000 cells/well in a plate containing 30% FBS in complete medium, and cultured at 37 °C, 5% CO2. The medium was changed every 3 days and the cell status was observed. After 2 weeks culturing, photographs were taken when individual cloned cells were large enough to be observed. Then it was stained with 0.1% crystal violet.

Migration and invasion assay

Matrigel was diluted with a serum-free DMEM high-glycemic culture medium, uniformly coated in the Transwell chamber, and incubated overnight for matrigel flooring. 100 μL cell suspension containing 5 × 105 cells was added to the Transwell chamber for 6 h adherence. After 24 h incubation according to the grouping, matrigel and bottom cells of the upper chamber were washed off, fixed with paraformaldehyde, washed with PBS, stained with crystal violet, then photographed and counted the number of cells migrated and invaded. It was repeated three times.

Flow cytometry (FCM) assay

The cells in the logarithmic phase were seeded in 6-well plates and grouped into Control group and RAC groups (1, 5, 10 mg/mL RAC) or Control group, 10 mg/mL RAC group, 3-MA group and 10 mg/mL RAC + 3-MA group. 3-MA (S24823) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd (China). After 24 h of treatment, the cells were collected and the cell concentration was adjusted to 1 × 106 cells/mL. 500 μL binding buffer was added and centrifuged to discard the supernatant, and then 100 μL binding buffer was added and mixed. 5 μL Annexin V-FITC and 10 μL PI (556547, BD Pharmingen, USA) were added respectively and reacted at room temperature for 15 min away from light. Finally, 400 μL binding buffer was added, and the apoptosis rate was detected by a flow cytometer (C6, BD, USA).

Cell cycle assay

The cell suspensions in the logarithmic phase were inoculated into plates, and the culture plates were pre-cultured for 24 h. Then the experiment was carried out according to different doses of RAC (0, 1, 5 and 10 mg/mL RAC). After 24 h of administration, the cell cycle of the cells was detected by flow cytometry.

Transmission electron microscope assay

The cells were fixed in glutaraldehyde solution and washed 4 times with 0.1 M pH7.0 PBS. It was then fixed with osmic acid and rinsed again. Cells were dehydrated with gradient concentrations of ethanol solution, then treated with 100% ethanol, and then treated with pure acetone. Subsequent gradient permeabilization was performed by using an embedding medium, and then the cells were placed in a 0.5 mL dry tube (pre-filled with about 300 μL of embedding medium), and the polymerizer was heated at 70 °C overnight. The sections were sliced into 70 nm slices by using an ultrathin microtome (EMUC7, Leica, Germany), stained with 100 μL of uranyl acetate 50% ethanol saturated solution for 20 min, rinsed with 100 μL of lead citrate in double distilled water and stained for 15 min, and finally photographed.

AOPI assay

The AOPI staining was used to detect the effect of the combination of RAC and 3-MA on autophagy in RCC cells. The RCC in the logarithmic phase were placed on the coverslip, and the cells were divided into the Control group, 10 mg/mL RAC group, 3- MA group and 10 mg/mL RAC + 3-MA group and cultured in a cell incubator of 37 °C, 5% CO2 for 24 h. It was rinsed 3 times through PBS, dripped with freshly prepared 1 mg/L AOPI staining solution (CA1143, Solarbio, China), incubated at 37 °C, aspirated the AOPI staining solution, and rinsed 3 times with PBS. The slides were mounted with 50% glycerol and placed under an inverted fluorescence microscope to observe the acidic autophagic vesicles.

Western blot

Firstly, the total protein in RCC was collected and the BCA kit (pc0020, Solarbio, China) was used to detect the total protein. The 10% SDS-PAGE electrophoresis and transfer membrane were performed. The PVDF membranes were blocked with 5% skimmed milk powder for 1.5 h followed by washing with TBST, then it were put into the primary antibody diluent (5% BSA as the diluent) of LC3A/B, Beclin 1, SQSTM1/P62, E-cadherin, Vimentin, Bcl-2, Bax, Cyclin D1, Cleaved-Caspase 3, Pro-Caspase 3, Phospho-PI3K, PI3K, Phospho-pan-AKT1/2/3, AKT2, Phospho-mTOR, mTOR, Phospho-P38 MAPK, P38 MAPK, p-ERK1/2, ERK1/2, β-actin and GAPDH and incubated in 4 °C for 12 h. Then the membranes were washed with TBST and the secondary antibody IgG (H + L) HRP was incubated for 2 h. The ECL was used to detect protein bands, and the protein gray value was calculated by Image J. The details of the antibodies used are list in Table 1.

Table 1 Antibodies used for Western blotStatistical analysis

SPSS 20.0 was used for data analysis. One-way ANOVA analysis of variance is used to measure data across multiple groups, and the Tukey or Dunnett’s T3 test is used for comparison between groups. While the Kruskal–Wallis H test is used when the data was not normally distributed. All data were expressed as mean ± standard deviation (SD). P < 0.05 suggested that the difference was statistically significant.