Cell culture
The gastric cancer cell lines (AGS and MGC803) were cultured in DMEM Medium (ThermoFisher Scientific, Cat: 11965092) containing 10% fetal bovine serum (Biological Industries, BISH0744), 1% penicillin/streptomycin (Invitrogen). HEK-293 cells were cultured in DMEM Medium supplemented with 10% FBS (Biological Industries, BISH0744) and 1%penicillin/streptomycin. The gastric cancer AGS and BCG803 cells were authenticated by short tandem repeats profiling (STR). STR profiling of our AGS cells was found to be 100% consistent with the STR data of the AGS from China Infrastructure of Cell Line Resources. MGC803 cell STR profiling data was not accessible in public databases including ATCC. Cells were regularly tested for mycoplasma using Lookout Mycoplasma PCR detection kit (MP0035, Sigma) and only used when negative.
RNA isolation and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted with RNeasy Plus Mini Kit (Tiangen, DP451) following the manufacturer’s specifications. Reverse transcription was performed using the HiScript II Q RT SuperMix (Vazyme, R223-01). qRT-PCR was carried out using SYBR qPCR Master Mix (Vazyme, Q511-02) and 7500 Fast Real-Time PCR System (Applied Biosystems, Singapore). The 36B4 was used as an internal control. The sequence of the primers for qPCR was listed as follows: 36B4 F: GGC GAC CTG GAA GTC CAA CT; R: CCA TCA GCA CCA CAG CCT TC. CTGF F: CTC GCG GCT TAC CGA CTG; R: GGC TCT GCT TCT CTA GCC TG. CYR61 F: AGC AGC CTG AAA AAG GGC AA; R: AGC CTG TAG AAG GGA AAC GC. DUB1 F: GAG GCC GGG GCT CTG A; R: ACT GGG ATG TGC AGA CTT GG. TAZ F: AGA GTC GGG TCG GGA TTT GT; R: AGG CCG GAT TCA TCT TCT GGG
Plasmids and siRNA
The DUB1 and TAZ plasmid are acquired from HANBIO Company (https://www.hanbio.net). The DUB1 and TAZ deletion constructs were sub-cloned from the full-length plasmid. The HA-K48 and HA-Ub plasmids were used in previous study.. The Lipofectamin 2000 (1662298, Invitrogen) was used for the plasmids transfection. Small interfering RNAs were used for specific gene knocking-down. The DUB1 siRNA sequences were: (1) CCG GCA AGC UGC GAA UAU UTT; AAU AUU CGC AGC UUG CCG GTT, and (2) GCA CAC CAC UGA AGA GAU UTT; AAU CUC UUC AGU GGU GUG CTT. The negative control siRNA sequences were: UUC UCC GAA CGU GUC ACG UTT; ACG UGA CAC GUU CGG AGA ATT. The RNA iMAX reagent (13778150, invitrogen) was used for siRNA transfection. For lenti-virus based DUB1 silencing, shDUB1 was cloned into the vector pLKO.1, which was co-transfected with pMD2.G and psPAX2 envelop plasmids into HEK293 cells. The DUB1 shRNA lenti-virus was harvested after 48 hours. The gastric cancer cells were incubated with 2 ml antibiotic-free medium containing 200 ul lentivirus.
Western blot
Standard western-blot assays were utilized to analyze the relative protein expression in cells. The following antibodies were used for western blot assays: anti-Flag-M2 (A8592, Sigma, 1:1000), anti-HA (2013819001, Roche, 1:1000) anti-Myc (9E10, Santa Cruz, 1:1000), anti-GAPDH (0411, Santa Cruz, 1:1000), anti-TAZ (Cell Signaling Technology, CST83699, 1:1000), anti-DUB1 (Sigma, HPA12082, 1:1000), Protein signals were detected with an ECL kit (Millipore Co., Billerica, Massachusetts, USA).
Luciferase reporter assays
For TEAD luciferase activity assays, the MGC803 and AGS with siDUB1 or siControl cells transfected with the TEAD luciferase reporter vector for 24 h. Cells were then harvested for assays. Luciferase reporter assays were performed using the dual luciferase assay kit (Promega). The pRL-null vector expressing renilla luciferase (Promega) was used as an internal control to normalize the transfection efficiency.
Wound healing and Transwell assays
For the wound-healing assay, the MGC803 and AGS with siDUB1 or siControl cells were seeded in a 6-well plate until confluent and then wounded with a sterile tip. The cells pictures were acquired at the indicated time points after scratching. The distances between the two edges of the scratched wound were measured using Image J software. The trans-well system (8 μm pore size, Corning) was employed for cell migration and invasion assays. For invasion assays, the upper chambers were coated with matrigel (BD Biocoat, USA). After 24 h, the gastric cancer cells that had migrated through to the bottom of the insert membrane were fixed, stained with crystal violet and counted under ×20 objective lens. The experiments were in three repeats.
Cycloheximide assay
MGC803 cells were transfected with siRNA or siControl for 24 hours. After that, the Cycloheximide was added into culture medium with the final concentration of 100 μmol/L. Cell lysis were collected at 0, 3, 6 and 9 hours after the treatment of cycloheximide. For HEK293 cells, the cells were transfected with 2ug Flag-DUB1 or Flag vector. After 24 hours, cells were treated with cycloheximide with final concentration of 100 μmol/L. Cell lysis were collected at 0, 3, 6 and 9 hours after the treatment of cycloheximide.
Immunofluorescence (IF) staining
The MGC803 Cells on the coverslips were fixed with 4% paraformaldehyde and incubated with the primary antibody against DUB1 (Sigma, A300-940A), TAZ (CST, 71192) at 4 °C overnight. After that, the cells were washing with PBS. Then the cells were then incubated together with fluorescence-conjugated secondary antibody (Invitrogen, Carlsbad, CA). Finally, the cells were subsequently counterstained with DAPI (Life Technology). The staining Images were captured through the confocal laser-scanning microscope (Leica TCS SP8 STED). The fluorescence-integrated density was measured by ImageJ software.
Clone formation assays
The MGC803 and AGS were seeded in six-well plates overnight and treated with 50nM DUB1 siRNA or 50nM siControl. After twenty-four hours, the gastric cancer cells were washed with PBS, trypsinized and plated at low density (5000 cell/well in six-well plate). The cells were cultured for 10 days and the medium was refreshed every two or three days. The colonies were stained with crystal violet. The number of the clones in a given area was counted for each condition.
Co-IP assays
The co-immunoprecipitation assays were performed as previous described. The MGC803 total cell lysls were collected and pre-cleared with rabbit IgG for 2 h and subsequently immunoprecipitated with DUB1 antibody (Sigma, A300-940A) over night, while rabbit IgG (Santa Cruz) was used as the negative control. The bounded protein was analyzed by Anti-TAZ (CST, 71192). For the overexpression experiment, HEK293 cells were transfected with 5ug Flag-DUB1 (Full length or deletion domains) and Myc-TAZ (Full length or deletion domains) in 10 cm dish. Cell lysates were pre-cleared with IgG and subsequently incubate with anti-Flag-M2 (A8592, Sigma, 1:1000) antibody, while mouse IgG was used as the negative control. The bound proteins were analyzed by western blotting.
In vivo ubiquitination assays
For in vivo ubiquitination assays, cells were transfected with vectors, including expressing Myc-TAZ, Flag-DUB1 and HA-Ub for 24 h respectively. Cells were then treated with MG132 (10 μM) for 6 h, and the levels of Myc-TAZ ubiquitination was determined by IP with anti-HA antibody (2013819001, Roche,1:1000) followed by western-blot assays with an anti-Myc (9E10, Santa Cruz, 1:1000).
In vivo tumorigenesis assay
For in vivo tumorigenic experiment, MGC803 cells were infected with shControl virus or shDUB1 virus. After 48 hours of infection, cells were treated with 1ug/ml puromycin for 3 days. MGC803 cells (2×106) were injected into the right dorsal flank of 4-week-old female BALB/c nude mice. Tumor formation in nude mice was monitored over a 4-week period. The tumor volume was calculated by the formula: tumor volume = 0.5 × length × width2. The mice were sacrificed five weeks after injection. The mice were sacrificed and the tumors were weighted and photographed. The experiments were performed under the protocols approved by ethnic committee of Xinxiang Medical University.
Cell proliferation assay
MGC803 and AGS cells were transfected with siDUB1 or siControl in 24-well plates. Twenty-four hours after transfection, the cell number was countered and 4000 cells were seeded into 96-well plates. The relative cell viability was measured at the indicated time points. Cell numbers determined determined using CCK8 cell proliferation reagent by measuring the absorbance at 450 nm. Cell proliferation was further analyzed by EdU incorporation and flow cytometry. Gastric cancer cells were determined by using the 5-ethynyl-20-deoxyuridine (EdU) assay kit (Ribobio, Guangzhou, China). For quantification analysis of the images, each data point represents the positive fluorescence area calculated from a minimum of five randomly chosen fields from three individual experiments. EdU incorporation FACS assay was performed according to the manufacturer’s instructions. The experiments were performed in triplicate. For cell cycle analysis, MGC823 cells were transfected with 50 nM siDUB1 or siControl. After 24 hours, the cells were fixed with 70% ethanol and stained with propidium iodide. Twenty-four hours post transfection, the cells were stained with propidium iodide and annexin V. A BD LSR FACS was used to measure the fluorescence intensity.
Tissue microarray (TMA) and immunohistochemistry (IHC)
One hundred paraffin-embedded gastric cancer samples were acquired from Shanhai OTUDO Biotech Company (http://www.superchip.com.cn). All the gastric tumor samples were examined with pathological specialists. The pathological grade plus lymph node metastasis status of each sample were acquired from Shanhai OTUDO Biotech Company. The usage of the samples was approved from the Shanhai OTUDO Biotech Company with written informed consent from all the patients. Specific antibodies against TAZ (CST, 71192) and DUB1 (Sigma, HPA12082) were used to detect the staining density in human samples. The scores were calculated on the intensity and percentage of positive tumor cells in the whole tissue, which were evaluated according to the Fromowitz Standard. The staining intensity was graded as: no staining, 0; weakly positive, 1; moderately positive, 2 and strongly positive, 3. The percentage for positive cells was into four grades: 0-25% staining, 1; 26-50% staining, 2; 51-75% staining, 3 and 76-100% staining, 4. The staining 1-2 was regarded as low expression, while the stain 3-4 was regarded as high expression. All staining were assessed at 200X magnifications and at least three fields from each core were counted.
RNA sequencing and data analysis
The global gene expression analysis (siControl and siDUB1) was based on RNA sequencing platform from BGI (Beijing Genomic Institute). The RNA sequence data are deposited in the Gene Expression Omnibus (GEO) database (Assessing number: GSE143947). Analysis was performed for differentially expressed genes (P < 0.01 and fold change > 2) by Ingenuity Pathway Analysis (IPA). For gene set enrichment analysis of RNA-seq data, gene sets of Conserved Hippo Signature were used and downloaded from Molecular Signatures Database v7.4, GSEA was implemented using the GSEA 4.1.0 software, with default parameters. Volcano plot was generated using ‘ggplot2’ package in R (threshold P<0.05 and fold change>1.5).
Analysis of TCGA data and Progression-free survival data analysis
Gene expression data for 385 TCGA gastric cancer patients were downloaded from the webpage (http://gepia.cancer-pku.cn/index.html). The expression of DUB1 mRNA level between normal gastric tissue and different gastric cancer stages were generated from GEPIA online software. The progression-free survival (PFS) survival data of TAZ and DUB1 were generated from KMPLOT online analysis database (https://kmplot.com). The gene affy ID was 202133_at for TAZ and 227093_at for DUB1. The PFS survival data of DUB1 and TAZ in gastric cancer patients were generated from KMPLOT database.
Statistical analysis
No specific statistical tests were used to predetermine the sample size. Statistical analysis was performed using GraphPad Prism 7 software or SPSS version 23.0. Data are expressed as the mean ± s.e.m. values. Differences between two independent groups were evaluated with Student’s t-test. The Kaplan−Meier method with the log-rank test was applied for survival analysis. Differences were considered to be statistically significant when P < 0.05 (*P < 0.01; **P < 0.001).