HK-2, 786-0, A498, ACHN, CaKi-1, and CaKi-2 cells were purchased from Shanghai Biology Institute (Shanghai, P.R. China). Cells were maintained in DMEM media (Trueline, Kaukauna, WI, USA) containing 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA), L-glutamine (2 mM) and 1% penicillin-streptomycin (Solarbio; Beijing, P.R. China). Cells were placed in a 37°C incubator with 95% air and 5% CO2. The NF-κB inhibitor was dissolved in DMSO (D2605, Sigma, USA).
Total RNA was extracted using TRIzol (Invitrogen, Waltham, MA, USA). cDNA was generated using a cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). Real-time PCR was performed as follows: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 45 seconds. Β-actin was the internal control. The 2−ΔΔCt method was used for gene expression. All experiments were performed using three replicates. The primer sequences were as follows: F: 5′-GAGCAAATCCAGAACCAG-3', R 5′-TCAAACTCCCAAACAATC-3′; GAPDH: F 5′-AATCCCATCACCATCTTC-3′， R 5′-AGGCTGTTGTCATACTTC-3′; nuclear factor kappa B subunit 1 (NF-κB), F 5′-CGGGGACGGTCTGAATGC-3′, R 5′-CTGCCAATGAGATGTTGTCGTG-3′.
Proteins were extracted in RIPA lysis buffer (JRDUN, Shanghai, P.R. China), in the presence of Protease Inhibitor Cocktail (EDTA-free; Roche, Heidelberg, Germany). The concentration was calculated by a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). A total of 25 μg of proteins were separated by 10% SDS-PAGE gels before being transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA) overnight. The membranes were blocked with 5% non-fat dry milk (1 hour at room temperature). Primary antibodies were incubated at 4°C overnight, followed by incubation of secondary antibody anti-mouse IgG (1:1,000; Beyotime, Shanghai, P.R. China) for 1 hour at 37°C. The bands were detected using an enhanced chemiluminescence system (Tanon, Shanghai, P.R. China). The primary used in this study were as follows: TRIM27 (#15099; CST, USA), NF-kB (#8242; CST, USA), cleaved caspase 3 (Ab32042; Abcam, UK), H3 (#4499; CST, USA), GAPDH (#5174; CST, USA).
Knockdown and overexpression of TRIM27
Lentiviral plasmids (pLKO.1) with three siRNAs targeting human TRIM27 (NM_006510.5) and a control siRNA (siNC) were designed and generated (Major, Shanghai, People's Republic of China). Lentiviral plasmids (pLVX-puro) with full length human TRIM27 or a mock plasmid (negative control) were generated and transiently transfected into Caki-1, Caki-2, and 786-0 cells using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Cells were harvested 48 h post-transfection. The TRIM27 siRNAs were as follows: siTRIM27-1: 941-959, CCCAGTTCTCTTGCAACAT); siTRIM27-2: 1052-1070, GGGCTGAAAGAATCAGGAT; and siTRIM27-3: 1495-1513, GGATTCTGGGCAGTGTCTT.
Cell Counting Kit-8 (CCK-8) assay kits (SAB, College Park, MD, USA) were purchased for the cell proliferation assay. Briefly, cells were transfected and seeded in 96-well plates for various time points. CCK-8 solution (1: 10) was incubated for 1 h before harvest. The optical densities were measured using a microplate reader (Pulangxin, Beijing, P.R. China) at 450 nm.
Caki-1, Caki-2, and 786-0 cells were collected and examined using an Annexin V-fluorescein isothiocyanate' apoptosis kit (Beyotime, Shanghai, P.R. China) 48 h post viral infection. Cells were examined by flow cytometry (BD, San Diego, CA, USA)
Cell extracts were incubated with different antibodies in the presence of Protein A/G beads (Santa Cruz Biotechnology, Dallas, TX, USA) overnight. The beads were washed thoroughly and the elutes were examined using SDS-PAGE and Western blot.
siNC or siTRIM27 was transfected. Cells were lysed in RIPA buffer (1% SDS) by sonication. Lysates were incubated with Protein A/G PLUS-agarose (sc-2003; Santa Cruz Biotechnology, USA) for 1 h. IgG (sc-2027; Santa Cruz Biotechnology, USA) was added to all samples and incubated overnight at 4℃. Nuclear pellets were collected following centrifugation at 3000 rpm at 4℃, followed by washing with Protein A/G Plus-Agarose beads four times. Proteins were loaded onto 4%–20% gradient SDS-PAGE gels. Signals were detected by anti-IκBα antibody (#4812; Cell Signaling Technology, Danvers, MA, USA) and anti-ubiquitin antibody (ab7780; Abcam, UK).
All animal procedures were performed according to the Institutional Animal Care and Use Committee. Experiments were approved by the independent ethics committee of the Changhai Hospital, Naval Medical University, Shanghai. siNC- or siTRIM27-transfected Caki-2 cells (n = 2 × 106), and oeNC or oeTRIM27-transfected Caki-1 cells (n = 2 × 106) were injected into the right flank of nude mice (4–6 weeks old, n = 6 in each group; Shanghai Laboratory Animal Company, Shanghai, PR China). Mice were sacrificed by cervical dislocation after 6 weeks. Mouse tissues were collected in 4% formalin.
TUNEL assays were performed on sections using an ApopTag kit (11684817910; Roche, Switzerland) principally according to the supplier's instructions. Three replicates were required for each sample.
Tissues were fixed in 10% formalin for 48 h before embedding in paraffin. Tissues were processed into slices using a microtome (Leike, China). A series of xylene and alcohol incubations were performed. Tissues were observed by hematoxylin and eosin staining.
Tissues were embedded in paraffin. Slices (5 μm) were treated in H2O2 (3%) dissolved in methanol and 5% normal horse serum before staining. Sections were incubated with the following primary antibodies overnight at 4°C: anti-TRIM27 antibody (ab137638; Abcam, UK) and anti-NF-KB antibody (ab16502; Abcam, UK). The sections were then incubated with secondary goat anti-rabbit IgG (1:100; ab64256; Abcam) for 30 min at room temperature. The sections were visualized with diaminobenzidine and hematoxylin. Cells with >25% positive staining were defined as having “high expression.”
Analysis was performed by GraphPad Prism Version 7.0 (La Jolla, CA, USA). Data are shown as mean ± SD. One-way ANOVA was performed, and P-values < 0.05 indicated statistical significance.