Cell culture
Human bladder cancer cell lines (T24 and 5637), the human normal bladder epithelial cell line SV-HUC-1, and the human kidney epithelial cell line 293T were obtained from the National Collection of Authenticated Cell Culture (Shanghai, China). The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C under 5% CO2. Additionally, we performed PCR-based Mycoplasma contamination testing of the cell cultures.
Clinical samples tissue microarray (TMA)
Two bladder cancer tissue microarrays (HBlaU060CS02 and HBlaU079Su01) were obtained from Shanghai Outdo Biotech (Shanghai, China). The study of TMA slides was conducted with ethical approval from the Clinical Research Ethics Committee, Outdo Biotech (Shanghai, China).
Immunohistochemistry (IHC) staining
Immunohistochemistry staining was performed on the HBlaU060CS02 and HBlaU079Su01 tissue microarrays using standard procedures. The sections were incubated overnight at 4 ℃ with a primary antibody against DGKZ (anti-DGKZ, 1:1000, ab239081, Abcam) and visualized using a DAB detection kit. Hematoxylin counterstain was used and the sections were observed using Aperio Digital Pathology Slide Scanners. The expression of DGKZ in tumor cells was semi-quantitatively assessed by scoring the percentage of positively stained cells (0-4: 0 <1%, 1 1-5%, 2 6-25%, 3 26-50%, 4 >50%) and the staining intensity (0-3: 0 negative, 1 weak, 2 moderate, 3 strong).
Cell growth, apoptosis, wound-healing and invasion assay
Cell proliferation was assessed using the Celigo cytometry system and MTT assay (3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenylte-trazolium bromide), following the manufacturer’s instructions. Cell apoptosis was detected by flow cytometry analysis (Millipore, USA) after staining the cells with Annexin V-APC (eBioscience, USA). For the wound healing assay, cells were scratched when they reached almost 100% confluence in 6-well plates. After 24 hours, the scratched areas were photographed, and the distance between the edges was measured using the ImageJ software. Transwell assays were performed according to standard procedures to assess the cell invasion ability.
Plasmid tansfection and lentivirus infection
To investigate the functional role of DGKZ in bladder cancer, we performed plasmid transfection and lentiviral infection experiments. The human DGKZ shRNA sequence (5’-CTCTGAAAGCAAGCAAGAA-3’) was designed and cloned into the GV493 vector (GeneChem, China). The GV493 vector, along with lentivirus packaging plasmids (pHelper 1.0 and pHelper 2.0), were transfected into 293T cells. Lentiviral particles containing DGKZ shRNA were generated and added to the cultured BCa cells in the presence of polybrene. To establish stable cell lines, the BCa cells were treated with puromycin (2.0 μg/mL) for to 4-5 passages. For the overexpression experiments, full-length DGKZ was amplified from human cDNA and cloned into the GV491 plasmid (GeneChem, China). Plasmid transfection and lentivirus infection were performed using the same procedure as described above.
RNA isolation and quantitative reverse transcription and real-time quantitative PCR (qRT-PCR)
Total RNA was isolated from clinical tissues and cells using TRIzol reagent (Invitrogen, USA). qRT-PCR was performed using the Power SYBR Green master mix (Roche). DGKZ mRNA expression levels were quantified using the following primer sequences: forward 5’-AGCAAGCAAGAAGAAGAAGAGG-3’ and reverse 5’-GGATTGAGATACCAGAGGAAAGAC-3’. The housekeeping gene β-actin (ACTB) was used as an internal control, and its expression was detected using the following primer sequences: forward 5’-GCGTGACATTAAGGAGAAGC-3’ and reverse 5’-CCACGTCACACTTCATGATGG-3’.
Western blot
Clinical tissues and cells were harvested and lysed in RIPA buffer (Solarbio, China). Protein samples (40 µg) were separated by 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, USA). The Membranes were blocked with a solution containing 5% non-fat milk and incubated overnight at 4°C with specific primary antibodies against DGKZ (1:500, No. SAB1300449, Sigma) and β-actin (1:5000, No. Sc-69879, Santa Cruz). The detailed procedure was described in our previous study (12).
Co-IP and LC-MS/MS
To identify potential interacting proteins with DGKZ, Co-IP and LC-MS/MS assays were performed. Flag-vector and Flag-DGKZ plasmids were transfected into 293T cells for 24 hours, followed by immunoprecipitation. The Co-IP assay was performed using a Pierce Crosslink Magnetic IP/Co-IP Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Whole-cell lysates were incubated with the desired antibodies, and the target protein was pulled down with protein A/G Magnetic Beads overnight at 4°C. The beads were then washed, and the bound proteins were eluted. After Co-IP, equal amounts of proteins were loaded onto 10% SDS-PAGE gels and stained with Coomassie blue G250 (BioRad, USA). The protein bands were excised, and the immunoprecipitates were analyzed by LC-MS/MS. Raw data were imported into Proteome Discoverer 2.2 (Thermo Fisher Scientific) for protein identification using Mascot 2.6 engines. Protein digestion was performed with trypsin at 37°C overnight, and the digested proteins were desalted for LC-MS/MS analysis (Thermo Scientific, USA). The antibodies used for Co-IP were anti-Flag (1:2000, F1804, Sigma), anti-RB1 (1:1000, 10048-2-1g, Proteintech), anti-STAT3 (1:1000, 9139, CST), anti-HNRNPM (1:1000, ab177957, Abcam), anti-CTNNB1 (1:1000, 8480, CST), anti-CSNK2A2 (1:1000, 10606-1-AP, Proteintech), anti-CEBPZ (1:1000, 25612-1-AP, Proteintech), anti-STAT1 (1:1000, ab31369, Abcam), anti-MAPK1 (1:1000, ab32537, Abcam), anti-PTPN3 (1:1000, NBP2-93945, Novus), and anti-IgG (1:10000, No. 7076, CST).
RNA sequencing
Total RNA samples (1 µg per sample) were used as input material for RNA sample preparation. Sequencing libraries were generated using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations. Unique index codes were added to each sample to attribute the sequences. PCR products were then purified using the AMPure XP system, and library quality was assessed using the Agilent Bioanalyzer 2100 system. After cluster generation, the library preparations were sequenced using an Illumina Novaseq platform, generating 150 bp paired-end reads.
Animal experiments
Four-week-old male BALB/c nude mice were purchased from Shanghai Lingchang Biotechnology Co., Ltd. (Shanghai, China) and were housed under sterile and pathogen-free conditions. Mice were divided into two groups (10 mice per group). Lentivirus-transduced T24 cells (1×10^7 cells) stably expressing DGKZ-shRNA or control cells were subcutaneously injected into the right armpit of the nude mice. Tumor burden was monitored by measuring tumor volumes, and the mice were euthanized with 1% isoflurane inhalation before being sacrificed by cervical dislocation, four weeks after cell injection. All animal procedures were approved by the Ethics Committee of the Second Hospital of Tianjin Medical University.
Statistical analysis
All data are presented as the means ± standard error of the mean (SEM) from three independent experiments using SPSS software (version 17.0). Differences between groups were assessed using two-tailed Student’s t-tests or one-way analysis of variance (ANOVA). The clinical and pathological characteristics of the patients were collected and analyzed accordingly. Qualitative variables were compared using the chi-square (χ2) test, and quantitative variables were compared using t-tests. Survival analysis was conducted using the Kaplan-Meier method, and Cox proportional hazards models were used for univariate and multivariate analyses. Spearman’s correlation analysis was performed to assess the correlation between the two variables. A p-value ≤ 0.05 was considered statistically significant.