Cell culture
The human bladder urothelial carcinoma cell lines, HT-1376, UM-UC-3, T24, J82, 5637, RT4, along with human immortalized uroepithelium cell line SV-HUC-1 and human embryonic kidney cell line 293T were obtained from American Type Culture Collection. EJ cells were purchased from the Institute of Biochemistry of Chinese Academy of Science. Cells were cultured in different media (EMEM for HT-1376 and J82; DMEM for UM-UC-3, 293T and EJ; RPMI-1640 for T24 and 5637; McCoy’s 5A for RT4; F12K for SV-HUC-1) supplemented with 10% of fetal bovine serum (ExCell bio, China) and 1% of penicillin/streptomycin (Thermo Fisher, USA) at 37°C with 5% of CO2.
Western blot
Cells were lysed with RIPA buffer (Beyotime, China) supplemented with 1% protease/phosphatase inhibitors (CWBIO, China) and the concentration of protein was calculated using PierceTM BCA Protein Assay Kit (Thermo Fisher, USA). Equal amounts of samples were loaded into 10% SDS-PAGE gels and then transferred onto PVDF Western Blotting Membrane (Roche, Switzerland). Membranes were incubated with specific primary antibodies overnight after blocked with 5% skimmed milk powder for 1 h. Primary antibodies used were anti-ANAPC11 (14090, CST, USA), anti-GAPDH (AC001, Abclonal, China), anti-FOXO3 (NBP2-16521, Novus Biologicals, USA), anti-HA (51064-2-AP, Proteintech, China), anti-ubiquitin (YM3636, ImmunoWay, USA), anti-p21 (2947, CST, USA) and anti-GULP1 (19902-1-AP, Proteintech, China). Next day, membranes were incubated with HRP conjugated secondary antibodies (Goat Anti-Rabbit IgG and Goat Anti-Mouse, CWBIO, China), visualized and captured by SmartChemeTM (SAGE, China). The intensity of blots was quantified via ImageJ software.
Patients and samples
A cohort of 110 UBC patients who underwent surgery at Sun Yat-sen Memorial Hospital, Sun Yat-sen University was included in this study. Informed consent was obtained from each patient. The pathological diagnosis of all patients was confirmed and the clinicopathological characteristics of patients were summarized in Table 1. This study was approved by the Ethics Committees of Sun Yat-sen Memorial Hospital, Sun Yat-sen University.
Immunohistochemistry (IHC)
Paraffin-embedded tissues were rehydrated and antigen-retrieved before incubating with anti-ANAPC11 (NBP1-78050, Novus Biologicals, USA), anti-FOXO3 (2497, CST, USA), anti-p21 (2947, CST, USA) or anti-GULP1 (19902-1-AP, Proteintech, China) at 4°C overnight. After developing with horseradish peroxidase-conjugated secondary antibodies, the specimens were stained with diaminobenzidine (DAB) and hematoxylin.
Transfection, cycloheximide (CHX) and MG132 assays
For transient transfection, small interfering RNAs (siRNAs) (GenePharma, China) were synthesized and transfected via Lipofectamine RNAiMAX (Invitrogen, USA). The open reading frame (ORF) of ANAPC11, FOXO3 (HA-labelled) or NC was cloned into pcDNA3.1 plasmids and the transfection of plasmids was achieved by X-tremeGENE (Sigma, USA). For stable transfection, short hairpin RNA (shRNA) specifically targeting ANAPC11 plasmid was constructed using the pLVX-shRNA2-Puro frame. The CRISPR-Cas9 mediated knockout of ANAPC11 vector was produced following Zhang’s protocol using lentiCRISPR v2 frame[22]. ShRNA or CRISPR vector was transfected into 293T cells along with psPAX2 and PMD2.G plasmids to package virus. Cells were infected with lentivirus and selected with puromycin. The sequences of siRNAs, shRNA, and small guild RNA (sgRNA) were listed in Table 2.
CHX or MG132 was supplemented to the medium to achieve a final concentration of 30 μg/mL or 20 μM, respectively. Cells were collected at indicated time point for CHX assays or 48 h after MG132 manifestation.
The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, clonal formation assay, flow cytometry for cell cycle detection, wound healing, Transwell cell migration and invasion assays
MTS assay, clonal formation assay and flow cytometry, wound healing, Transwell cell migration and invasion assays were conducted as previously described[29].
Immunoprecipitation (IP) and mass spectrometry
UBC cells with different treatment were collected and lysed with Cell lysis buffer for WB and IP (APE×BIO, USA). After centrifugation, the supernatant was quantified and equal amount of protein was incubated with Protein A/G magnetic beads (Biolinkedin, China) conjugated with specific antibodies at 4°C overnight. Beads were washed and precipitated protein was eluted and subjected to western blot and mass spectrometry. Mass spectrometry was performed by the Bioinformatics and Omics Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University.
Glutathione S-transferase (GST) pull-down
The coding sequence of ANAPC11 was cloned into pGEX-6P-3 vector (IGEbio, China). The plasmid was transformed in E.coli and induced by 1 mM IPTG for expression of GST-ANAPC11 fusion protein. To purify the fusion protein, glutathione sepharose beads (Abcam, UK) were administrated according to manufacturer’s instruction. The fusion protein was further incubated with T24 or UM-UC-3 cell lysate and the eluted complex was detected by coomassie blue staining and western blot analysis.
RNA isolation, reverse transcription, real-time PCR (RT-PCR) and Chromatin immunoprecipitation (ChIP)
RNA was extracted using RNA-Quick Purification Kit (ES Science, China). Thereafter, 1 μg of total RNA was reverse transcribed with PrimeScript RT Master Mix (Takara, Japan) following manufacturer’s instruction. RT-PCR was performed with Hieff UNICON ® Power qPCR SYBR Green Master Mix (YEASEN, China) on QuantStudio Dx (Applied Biosystem, USA). CHIP was conducted using Pierce Magnetic ChIP Kit (Thermo Fisher, USA). Briefly, cells were subjected to 1% formaldehyde for crosslinking. Then cells were lysed, MNase digested and sonicated to obtain DNA fragments. After immunoprecipitation with anti-FOXO3 antibody (NBP2-16521, Novus Biologicals, USA), outcomes were Proteinase K treated and samples were recovered with DNA Clean-Up Column. Primers used were listed in Table 3.
Dual-luciferase reporter assay
Promoter region (-2000 bp to -1 bp, upstream of transcription start site (TSS)) sequences of CDKN1A and GULP1 were cloned into pGL3-basic vector separately. Then pGL3-basic, pRL-TK and pcDNA3.1 with NC or FOXO3 ORF were co-transfected into 293T cell. 48 h after transfection, luminescence of firefly and Renilla was detected using Dual-Glo® Luciferase Assay System (Promega, USA). Relative firefly luminescence was normalized to Renilla luminescence.
Animal experiments
Four-week-old male BALB/c nude mice were randomly divided into control and experimental group (n = 5 for each group). For subcutaneous xenograft model, 5 × 106 ANAPC11-NC or -KO (knockout) T24 cells in 100 μl PBS were injected into left flank of the each mouse and the tumor size was measured every 5 days following the formular volume (mm3) = 1/2 × (length) × (width)2. Mice were euthanized 30 days after injection and tumor sections were fixed, embedded, hematoxylin and eosin (HE) and IHC stained. For lymph node metastasis model, control and ANAPC11 knockout cells were infected with Lenti-luciferase P2A-Neo and selected with G-418 (1 mg/ml). 5 × 106 T24 cells in 50 μl PBS were administrated into the right footpads of mice. Mice were intraperitoneal injected luciferin and 10 min later anesthetized with 3% isoflurane. The bioluminescence was detected and captured via AniView 600 (BLT, China). The procedure of animal experiments were evaluated and approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University in compliance with the guide for the Care and Use of Laboratory Animals.
Statistical analysis
Data of at least three independent experiments were analyzed by SPSS 20.0 (IBM SPSS Statistics, USA) and were presented as mean ± standard deviation (SD). Student’s t-test, Chi-square test, Wilcoxon rank-sum test, Mann-Whitney U test, one-way analysis of variance (ANOVA) were applied to compare difference among groups based on data type. Log-rank test was conducted in Kaplan–Meier survival analysis to assess patients’ prognosis. P < 0.05 was considered as statistic significant.