4.1 TMA and tissue samples
In this study, we used TMs containing 80 tumor tissues and 80 adjacent normal tissues obtained from GC patients (HStmA180Su13; Shanghai Outdo Biotech, Shanghai, China). An anti-CHRDL2 antibody was used to stain them, the staining intensity was scored and the CHRDL2 staining score was calculated. All 12 pairs of tumor and normal tissues were obtained from Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. All patients mentioned in this study had not received therapy, and the clinical stage for each section was obtained according to the TNM stage of American Joint Committee on Cancer and International Union Against Cancer( AJCC/UICC) for GC, 8th edition20,21.
4.2 Cell culture and experiments
Gastric-adenocarcinoma cell lines HGC-27, MGC-803, SGC-7901, HS-746T, BGC-823, and MKN-45, and the human immortalized gastric cell line GES-1 were obtained from the Cell Bank of the Chinese Academy of Sciences. We cultivated cells in Dulbecco's Modified Eagle Medium or RPMI-1640 medium containing 10% fetal bovine serum (Gibco Waltham, MA, USA) and 1% penicillin-streptomycin in a humidified incubator with 5% CO2 at 37°C.
STK3 cDNA was synthesized and inserted into pCDNA3.1 plasmid (PPL; Nanjing, Jiangsu, China), while using an empty pcDNA3.1 plasmid as a control. To silence the expression of YAP, we generated siRNA-YAP and use siRNA-NC as a control by Genomeditech (Shanghai, China). The full-length CHRDL2 cDNA was inserted into a lentiviral GFP-Puro vector (PPL; Nanjing), and we also cloned a small hairpin RNA (shRNA) targeting CHRDL2 into the piLenti-shRNA-GFP-Puro vector by PPL (Nanjing). For transfection, we first transduced MGC-803, HGC-27, and AGS cells with the lentiviruses mentioned earlier for 24 h, and then we replaced the medium with 5 µg/mL puromycin (Beyotime Biotechnology, Beijing, China) after 48 h. Using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) to complete the transfection, we then collected the cells after 48 h of transfection.
4.3 RNA extraction and real-time quantitative polymerase chain reaction
First, we used TRIzol reagent (Vazyme, Nanjing, China) to extract the total RNA following the manufacturer’s protocol. Then, RNA samples were reverse transcribed into cDNA using a Reverse Transcription System (Toyobo, Osaka, Japan). Messenger RNA (mRNA) levels were determined using ChamQ Universal SYBR qPCR Master Mix (Vazyme) on an Applied Biosystems 7900HT sequence detection system (Applied Biosystems, Foster City, CA USA). Using the 2 − ΔΔCt method, the relative expression of mRNA levels was determined relative to GAPDH. All the primers used are presented in Supplementary Table S1.
4.4 Western blotting analysis
Using a standard protocol, the total protein extraction of GC cells was performed in RIPA lysis buffer (Solarbio, Beijing, China) supplemented with phenylmethylsulfonyl fluoride (PMSF; Solarbio) and a protease inhibitor cocktail (Roche, Basel, Switzerland). We then determined the concentration of protein by BCA assay (Pierce, Thermo Fisher, Waltham, MA, USA). Western blotting was performed following a previously published protocol. The candidate antigens were detected by antibodies (all antibodies are described in detail in Supplementary Table S2). All bands were captured using a Tanon imaging system.
4.5 Cell proliferation assays
We determined the rates of cell proliferation using an EdU Labeling Kit (Beyotime Biotechnology, Haimen, China) and a Cell Counting Kit-8 (CCK-8; Dojindo, Shanghai, China). All experimental procedure followed the manufacturers’ instructions. To conduct the colony formation assay, we put 1 × 103 GC cells/per well into six-well plates for cultivation for 14 days.
4.6 IHC staining
For subcutaneous tumors, we made formalin-fixed and paraffin-embedded specimens, and then cut these into 5 µm thick sections and fixed them on glass slides. These sections were then incubated with different antibodies. To determine the ratio of positive tumor cells, we performed semiquantitative evaluation by assessing the percentage of positive cells. The details are as follow: 0 = 5–25%, 1 = 26–50%, 2 = 51–75%, and 3 = ≥ 75%. We also evaluated the expression levels of CHRDL2, MST2, TAZ, Ki-67, and YAP by scoring the intensity of stained cancer cells as follows: 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellow), and 3 (strong staining, brown). Above all, scores ≥ 3 were regarded as high expression, and those < 3 were considered low expression.
4.7 Cell cycle analysis
We collected 5 × 105 cells in six-well plates, fixed with 75% ethanol, which were placed at 4°C overnight. Then, we resuspended the cells in 400 µL of PI/RNase Staining Buffer (Biosciences, NC, USA), and stained these cells for 30 min at room temperature in a dark room. Finally, we analyzed the cell cycle using flow cytometry.
4.8 Animal tumor transplantation
All animal experiments were approved by the local Laboratory Animal Ethics Committee of Ruijin Hospital (Shanghai, China). We purchased three- to four-week-old male BALB/c-nude mice from the Chinese Academy of Sciences (Shanghai, China) and divided them into two groups (n = 5 per group). Mice were subcutaneously injected with 5 × 106 MGC-803/CHRDL2 or MGC-803/vector cells. For measurements, we determined the tumor (a) length and (b) width every four days. The method of calculating tumor volumes was as follows: volume = 1/2 (a × b2). After 32 days, we killed the mice and harvested the tumors, which we stored at -80°C for further research.
4.9 Statistical analysis
We performed statistical analysis using SPSS 21.0 and GraphPad Prism 7.0. All data are shown as the mean ± SD from three or more independent experiments. Independent-sample t tests were performed between cell proliferation assay samples, qPCR samples, and for tumor weight and sizes between two groups. We estimated the correlation of CHRDL2 expression with the clinicopathological characteristics of GC using the Chi-square (χ2) test. Spearman’s correlation analysis was conducted to assess the association between CHRDL2, MST2, and YAP/TAZ in paraffin-embedded clinical tumor tissues. A P value < 0.05 was regarded as statistically significant (**p < 0.05; ***p < 0.01; ****p < 0.001).