Cell lines and cell culture

Renal clear cell carcinoma cell lines A498 (#CL-0254, Procell Life Science&Technology, Wuhan, China) and HUVEC (#CL-0122, Procell Life Science&Technology, Wuhan, China) were purchased from Procell Life Science&Technology and identified by short tandem repeat (STR) profiling. 786-O (SC0154) cells were purchased from the Yuchicell Biology Technology (Shanghai, China) and identified by STR profiling. The basic medium for 786-O cells was 1640 culture medium, for A498 cells was MEM culture medium, and for HUVEC was Ham’s F-12K culture medium. All of them were incubated at 37 °C in 5% CO2 and cultured in different kinds of basic medium added with 10% fetal bovine serum (FBS) (Newzerum, Christchurch) and 1% Penicillin streptomycin mixture (P/S) (#R20016, Biosharp, Guangzhou, China).

Chemicals and reagent

CDK4/6 inhibitors:Palbociclib (PD-0332991) HCl (#S1116), Proteasome inhibitors: MG132 (#S2619), mTOR inhibitors: Everolimus (#S1120), Bafilomycin A1 (BafA1), (#S1413) were purchased from Selleck (Shanghai, China). The shRNAs were purchased from GeneCopoeia (USA). The siRNAs were obtained from RiboBio (Guangzhou, China). The sequences of siRNA and shRNA are provided in the Table S1.

Immunohistochemistry (IHC)

Immunohistochemistry was performed with primary antibodies against RNF26 (#16802-1-AP, Proteintech, Wuhan, China 1:1000 dilution), TSC1 (#29906-1-AP, Proteintech, Wuhan, China, 1:1000 dilution), CDK4 (#11026-1-AP, Proteintech, Wuhan, China, 1:1000 dilution), p-S6K1 (Thr389) (#28735-1-AP, Proteintech, 1:1000 dilution), and VEGFA (#19003-1-AP, Proteintech, 1:1000 dilution) in the tissue microarray slides (#U081ki01, Bioaitech, Xian, China). The degree of binding was evaluated by multiplying the intensity score by the proportion of cells stained. The staining intensity scores were as follows: 0 (no staining), 1 (light yellow = weak staining), 2 (yellow brown = moderate staining), 3 (brown = strong staining).

Quantitative real-time PCR analysis

Total RNA was extracted from cells using TRIzol reagent (Termo Fisher Scientifc, USA). Reverse transcription was performed using Evo M-MLV reverse transcription premixed kit (#AG11728, Accurate Biology, Hunan, China), and qRT-PCR was subsequently performed using Evo M-MLV one-step RT-qPCR kit (#AG11732, Accurate Biology, Hunan, China), following the manufacturer’s instructions. β-actin was used as an internal control. The primer sequences (Bgi-write, Beijing, China) for RT‒PCR are provided in Table S2.

Western blotting assay

The protein was extracted with protein lysate (#G2002, Servicebio, Wuhan, China) added with protease inhibitor (#G2006, Servicebio, Wuhan, China) and phosphatase inhibitor (#G2007, Servicebio, Wuhan, China), and quantified using BCA Protein Detection Kit (#P0011, Beyotime, Shanghai, China). Lysate proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a 0.45 μm polyvinylidene fluoride membrane (Millipore, Billerica). After sealing with 5% skim milk, the membranes were incubated with corresponding primary antibody overnight at 4°C and secondary antibody for 1 h at room temperature. Protein signals were visualized using ECL detection reagent (Termo Fisher Scientifc, USA) and ChemiDoc XRS (Bio-Rad Laboratories, USA). The primary antibodies used were as follows: RNF26 (#16802-1-AP, Proteintech, 1:1000 dilution), TSC1 (#29906-1-AP, Proteintech, 1:2000 dilution), CDK4 (#11026-1-AP, Proteintech, 1:2000 dilution), S6K1 (#14485-1-AP, Proteintech, 1:4000 dilution), p-S6K1 (Thr389) (#28735-1-AP, Proteintech, 1:4000 dilution), VEGFA (#19003-1-AP, Proteintech, 1:2000 dilution), p-RB1(phospho S795) (#ab47474, Abcam, 1:500 dilution), GAPDH (#60004-1-Ig, Proteintech, 1:50000 dilution), and pRB1 S249/T252 (#701059, Thermo fisher scientific, 1:1000 dilution).

Transwell-invasion assays

Transwell-invasion assays were performed using 8-μm pore BioCoat Matrigel Invasion chambers (Corning, USA). The cells were starved with serum-free medium for 24 h. After spreading the matrix glue in the upper chamber of the chamber, the cells were digested into single cells with trypsin and re-suspended in the serum-free medium. Adding cells to the upper chamber according to the density of 6 × 104 cells per well, and the medium containing 10%FBS was added to the lower chamber. After 24 h of incubation, the cells were stained with 0.1% crystal violet for 30 min, and the unmigrated cells in the upper chamber were carefully removed with cotton swabs. Four visual fields were randomly selected under the microscope to count the cells passing through the membrane.

Colony formation assays

The cells were digested into single cells with trypsin and suspended in complete culture medium. The cell suspension was added to a 6-well plate at a density of 100, 200, 500 per well and cultured at 37 °C and 5% CO2. Replacing the culture medium in a week and abandoning the medium in two weeks. The cells were washed twice with PBS and fixed with paraformaldehyde for 20 min, and then stained with crystal violet for 30 min. Washing off the dye solution and counting the number of clones. Clone formation rate = (numbers of clones / number of inoculated cells) × 100%.

Angiogenesis assay in vitro

Matrix gel, sterile 24-well plate, pipette tips and other instruments used in the experiment were placed at 4 °C overnight the day before the experiment. The matrix glue was added to the 24-well plate at 250 μL per well with precooled pipette tips. The plate was incubated at 37 °C for 1 h until the matrix glue solidified. When the confluency of HUVEC reached 80%, they were digested by trypsin and resuspended with different tumor-conditioned supernatant. The cells were inoculated on the board containing matrix glue at 1 × 104 per well, and three replicates were made in each group. Cells were cultured and monitored for tube formation up to 48 h. Four visual fields were randomly selected for each well, and the number of tubes were counted and photographed.

Nude mouse xenograft assay

All animal experiments were examined and approved by the Institutional Animal Care and Use Committee (IACUC) of the Second Xiangya Hospital, Central South University (animal license number 20230474). Animal experiments are consistent with the guidelines for the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). BALB/C-nu/nu mice (6 weeks old) were purchased from SJA Laboratory Animal Company (Changsha, China). Since the sex of the mice had no effect on the results of the study, we chose half the males and half the females for the experiment. The mice were placed under standard conditions with a light / dark cycle of 12 h. They had easy access to food and water. Specific shRNA and shControl were used to transfect cells for 72 h, and puromycin was used to screen shRNA positive cells for 48 h. The cells were digested with trypsin and centrifuged by 1050 RPM for 5 min. After adding proper amount of PBS and fully mixing, the cell suspension was stored on ice and injected subcutaneously on the left side of the back of mice (5 × 106 cells per mice) as soon as possible (n = 5 mice per group). The length and width of the tumor were measured with vernier caliper every 2 days, and the tumor volume was calculated according to the formula (L × W2)/2. The mice were euthanized and the tumors were removed, photographed and weighed.

Immunofluorescence staining

786-O cells were inoculated on glass coverslips. Being fixed with 4% paraformaldehyde for 15 min, the cells were subsequently incubated with anti-RNF26 (#16802-1-AP, Proteintech, 1:200 dilution) or anti-TSC1 (#29906-1-AP, Proteintech, 1:250 dilution) antibody at 4 °C overnight, and washed several times with PBS. Then the cells were incubated with suitable fluorophore-conjugated secondary antibody for 1 h at room temperature in the dark. The slides were washed repeatedly with PBS and incubated with DAPI (#C1002, Beyotime, Shanghai, China) for 5 min. Images were captured using a fluorescence microscope.

Statistical analysis

The results were analyzed by using GraphPadPrism5 software. The sample size (N) of each statistical analysis is provided in the figure legends. The values between the two groups were compared by unpaired bilateral student’s t test, and multiple comparisons were made by one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test. In all cases, P < 0.05 was considered statistically significant, and the significance of differences was indicated as follows: *P < 0.05; **P < 0.01; and ***P < 0.001.