2.1 Ethics statement
2.2 Bioinformatic data analysis
A series of survival data, expression level data, clinical characteristics were obtained from ONCOMINE database (www.oncomine.org) by using the following search terms: ‘USP39’, ‘Cancer vs. Normal Analysis’, ‘Kidney Cancer’ and ‘mRNA’. Two datasets (Gumz Renal dataset and Jones Renal dataset) including RCC vs. normal kidney tissues were used to analyze the expression of USP39 in RCC and normal kidney tissues . One dataset (Zhao Renal dataset) with 176 RCC tissues was used to explore the effect of USP39 on survival analysis . Correlation analysis of USP39 and VEGF-A was performed on the Gumz Renal. All data are reported Log2 Median-Centered intensity in the Oncomine database.
2.3 Cell culture and treatments
A498, 769P, 786-O, ACHN, Caki-1, Caki-2, 293T and human umbilical vein endothelial (HUVEC) cells were purchased from the Cell Bank of Shanghai Academy of Life Sciences, the Chinese Academy of Sciences. Cells were cultured with 1640 or DMEM + 10% fetal bovine serum (FBS) + 1% penicillin double antibody at 37 °C and 5% CO2.
Recombinant plasmids pcDNA3-USP39 were constructed to overexpress USP39 in our laboratory. The two truncated forms of USP39, one with amino acids (AA) 1-100 (containing the RS-like domain, USP39 (1-100)) and the other with AA 101-565 (containing the ZnF, UCH1, and UCH2 domains, USP39 (101-565)), were acquired from the Key Laboratory of Cell Differentiation and Apoptosis of the National Ministry of Education (Shanghai Jiaotong University School of Medicine, Shanghai, China). Lentiviral USP39 knockdown plasmids (Lv-shUSP39, ShRNA sequences: 5’-GATTTGGAAGAGGCGAGATAA-3’) were prepared by Hollybio Biotechnology Company (Shanghai, China). Empty plasmids (Con) Lentiviral vector with nonspecific shRNA (Lv-shCon) were used as controls. Lentiviruses were constructed in 293T cells according to the manufacturer’s method. The knockdown and overexpression efficiency was assessed by RT-PCR and Western blot.
2.4 MTT assay
Cells with stable knockdown of USP39 were cultured in a 96-well plate at 2000 cells/well for Day 1, 2, 3, 4 and 5. At each time point, cells were incubated with 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MMT) solution for 4 h at 37°C and was terminated by acidic isopropanol solution. Half an hour later, cell viability was measured by 595 nm absorbance to acquire OD values. OD values of each day were used to draw the MTT growth curve. Each experiment was performed in triplicate.
2.5 Plate colony formation assay
Cells with stable knockdown of USP39 were cultured in a 6-well plate at 400 cells/well at 37°C for 14 days with the culture medium replaced at 3-day intervals. Cell colonies were fixed with 4% paraformaldehyde for 30 min and then stained under GIEMSA for 20min. Each cell colony was counted under a light microscope and photographed with a digital camera.
2.6 Flow cytometric assay and cell cycle detection
According to manufacturer’s instructions, cells were centrifuged, resuspended with PBS, fixed by addition of proof ethanol to a final ratio at 66%, incubated on ice for 15 min, resuspended in a working solution with 500ul Propidium Iodide (PI) buffer, 25ul PI (20x) and 5ul RNase A (50x), and incubated again at 37 °C for 40 min. Flow cytometric analysis was performed using a flow cytometer (BD Biosciences, USA). Red fluorescence was detected at the excitation wavelength 488nm, and the laser emission was detected at the same time.
2.7 Tubule formation assay
After 24-h serum-free culture, cell supernatants of 786-O and ACHN cells with knockdown or overexpressiom USP39 were collected. After addition of 50 μL Matrigel solution to each well of the 96-well plate, cells were incubated at 37°C for 1 h. HUVECs were resuspended with the collected cell supernatant or serum-free 1640 medium at 1 × 105 cells/mL after serum-starvation overnight. The resuspended HUVECs (100 μL) were added to the Matrigel-coated wells and incubated for 8 h at 37°C. Subsequently, HUVECs were imaged with an inverted fluorescence microscope (CKX41, Olympus, Japan). ImageJ was applied to analyze the number of meshes, the number of branches, and the tube length.
2.8 Quantitative real-time PCR (RT-qPCR)
Total RNA was extracted with Trizol reagents (Invitrogen) and cDNA was obtained using First-Strand cDNA Synthesis Kit (Invitrogen). The resulting cDNA was subjected to RT-qPCR with the indicated primer sets. RT-qPCR analysis was conducted by Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Relative gene expression was normalized to GAPDH with the 2-ΔΔCT assay. The primer sequences were used: for USP39, 5’-GCCAGCAGAAGAAAAAGAGC-3’ (forward) and 5’-GCCATTGAACTTAGCCAGGA-3’ (reverse); for VEGF-A, 5’- GCACATAGGAGAGATGAGCTTCC-3’ (forward) and 5’- CTCCGCTCTGAACAAGGCT-3’ (reverse) for β-actin, 5’-ATCGTGCGTGACATTAAGGAG-3’ (forward) and 5’-AGGAAGGAAGGCTGGAAGAG-3’ (reverse). Values were normalized to those of Actin.
2.9 Western blot
Cells with stable overexpression or knockdown of USP39 were lysed using RIPA lysis buffer (Beyotime, China), and the protein concentration was measured by BCA assay (Beyotime). Samples were prepared in SDS sample loading buffer, and transferred to the PVDF membrane. The main antibodies of Western blot were anti-USP39 (Abcam), anti-SRSF1 (Abcam), anti-SRPK1 (Santa Cruz), anti-VEGF 165b (R&D), and anti-VEGF-A (Abcam). The membrane was blocked by 5% milk (nonfat) in TBS (0.05% Tween 20) at room temperature for 1 h, and incubated with the primary antibodies for 2h and with HRP-conjugated IgG (rabbit). Western blotting detection system (Tanon) was applied to detect Chemiluminescence of the membrane.
2.10 Co-immunoprecipitation (CO-IP) analysis
Cells with stable overexpression or knockdown of USP39 were collected by RIPA buffer. Immunoprecipitation was conducted with anti-HA (Abcam) or anti-SRPK1 (Santa Cruz) or anti-Pan-phospho-SR (Santa Cruz). By incubation with protein A agarose (Santa Cruz), the antibodies were removed. Proteins were prepared and separated by 10% SDS-PAGE. The interaction between USP39 and SRPK1/SRSF1 was analyzed by Western blot using anti-Flag tag (Abcam) tag or anti-SRSF1 (Abcam).
2.11 Statistical analysis
All data were analyzed by SPSS version 22.0 (IBM corporation) and processed by GraphPad Prism 8.0. Independent sample t-test was used for comparison between groups. P < 0.05 was considered statistically significant.