Nci n87

The human GC cell lines AGS, SNU-1 and NCI-N87 were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in DMEM (AGS) or RPMI (SNU-1 and NCI-N87) supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100ug/ml) (all from Invitrogen, Carlsbad, CA). Cell lines were verified mycoplasma negative and identities were verified using the PowerPlex HS16 System kit (Promega).

Gastric cancer cell lines NUGC4 (JCRB0834) and NCI-N87 (ATCC^®CRL-5822) were cultured in Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal calf serum (FCS) and 1% penicillin/streptomycin, and maintained at 37 °C in a humidified atmosphere (5% CO₂). Cells were tested routinely for mycoplasma contamination. H. pylori strain G27 [31 (link)] was cultured on Wilkins–Chalgren (WC) Dent agar plates (OXOID, Hampshire, UK) in a microaerophilic atmosphere at 37 °C and 10% CO₂. Cells were infected at a multiplicity of infection (MOI) of 10 (OD₆₀₀ 1 = 2 × 10⁸ bacteria/mL) for 24 h and lysed in sodium dodecyl sulfate (SDS) buffer for protein expression analysis. IFN-γ (10 ng/mL) cells stimulated for 24 h were used as positive control.
Human peripheral blood mononuclear cells were isolated from H. pylori-negative healthy donors, after informed consent, by density gradient centrifugation with Pancoll (PAN-Biotech, Aidenbach, Germany). Cells were cultured with RPMI-1640 containing 10%FCS at 37 °C in a humidified atmosphere (5% CO₂) and infected with the H. pylori G27 strain at MOI 10 for 24 h. The supernatant was collected and co-cultured with gastric cancer cell lines (NUGC4 and NCI-N87) for 24 h. After co-culturing, gastric cancer cells were lysed in SDS buffer for protein expression analysis.

Cell lines of human breast cancer (SK-BR-3, BT-474, and MDA-MB-231), gastric cancer (NCI-N87), T-cell leukemia (Jurkat), and Human Embryonic Kidney cell 293 (HEK 293) were purchased from the China Center for Type Culture Collection (CCTCC). The Herceptin-resistant human breast cancer cell line (JIMT-1) was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). JIMT-1 and MDA-MB-231 cells were cultured in DMEM (Gibco), and all other cells were cultured in RPMI 1640 (Gibco) with 10% fetal bovine serum (Gibco). All cells were maintained at 37 °C in a 5% (vol/vol) CO₂ humidified incubator.
The B16 Mouse melanoma cell line (TCM2) was purchased from the Type Culture Collection of the Chinese Academy of Science (Shanghai, China). The B16-HER2 cell line was established by transfection with a vector (pcDNA3.4-TOPO, Invitrogen, A14697) that contained the full-length cDNA of hHER2 (acquired by RT-PCR from the mRNA of NCI-N87).
The BsAbs used in this study include M802 targeting hHER2 and hCD3, M806 targeting hHER2 and mCD3, MCO101 targeting fluorescein and hCD3, and MCO106 targeting fluorescein and mCD3. In addition, these BsAbs were constructed on the basis of a monovalent unit and a single chain unit, so this type of BsAb is collectively called MSBODY.

The AGS and NCI‐N87 GC cell lines were purchased from the American Type Culture Collection (MD, USA), and human HEK293T cells and the SGC7901, BGC823, and MGC803 GC cell lines were obtained from the Type Culture Collection of the Chinese Academy of Sciences (TCCCAS, Shanghai, China). MKN‐45 cells were obtained from the cell bank of the RIKEN BioResource Center (Tsukuba, Japan). SGC7901, NCI‐N87, BGC823, and MKN45 cells were cultured in RPMI‐1640 medium (Invitrogen Life Technologies, CA, USA), MGC803 cells were cultured in DMEM (Invitrogen Life Technologies, CA, USA), and AGS cells were cultured in F12K medium (Cellcook Biotech Co., Ltd., Guangzhou, China). The media for all the cell lines were supplemented with 10% fetal bovine serum (FBS; Gibco, CA, USA), 100 µg mL⁻¹ streptomycin, and 100 U mL⁻¹ penicillin (New Cell & Molecular Biotech, Suzhou, China), and cells were cultured in an incubator with 5% CO₂ at 37 °C. Cells were stored at −80 °C using CELLSAVING (New Cell & Molecular Biotech, Suzhou, China). All cells were tested negative for mycoplasma contamination and were authenticated based on STR fingerprinting before use.

GC cell lines AGS, HGC-27, MGC803, SGC7901, BGC823, SNU-1, NCI-N87, MKN-45 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and human non-malignant gastric mucosal cell line GES-1 was obtained from the China Center for Type Culture Collection (CCTCC). All the cell lines were cultured according to their protocols: AGS, HGC-27, MGC803, SGC7901, BGC823, SNU-1, NCI-N87, MKN45 were cultured in RPMI1640 medium with 10% fetal bovine serum sand 100u/ml penicillin/streptomycin, GES-1in DMEM medium with 10% fetal bovine serum and 100u/ml penicillin/streptomycin in cell incubator with 5% CO2 at 37℃.
Cells were transfected with 50nM miRNA mimics or 75nM inhibitor using lipofectamine 3000 (Invitrogen, USA). Interfering RNAs (siRNA) targeting SCIN was obtained from Genepharma (Shanghai, China) (siRNA1: 5'-GGUGAGAGCCACAGAAGUUTT-3'; siRNA2: 5'-GGAGCAGAGUAUGUAGCAATT-3'; siRNA3: 5'-GGACACCAAUUGUCAUCAUTT-3') and transfected at concentration of 50nM. Random sequence of RNA duplex was used as negative control for miRNA and siRNA. Plasmids expressing sh-SCIN or sh-NC were purchased from Genepharma (Shanghai, China).

Human gastric epithelial cell line GES-1 and 6 gastric cancer cell lines (MKN-28, MKN-45, AGS, BGC-823, NCI-N87 and SGC-7901) were obtained from American Type of Cell Collection (ATCC, Manassas, VA, USA) or RIKEN BioRe-source center (Ibaraki, Japan). GES-1, MKN-28, AGS, BGC-823, SGC-7901 and NCI-N-87 cells were cultured in RPMI 1640 medium (Invitrogen, Carsbad, CA, USA), MKN-45 cells were cultured in DMEM medium (Invitrogen), supplemented with 10% fetal bovine serum and incubated at 5% CO₂ incubator (Thermo) at 37 °C and 95% humidity. MLN4924 was purchased from ApexBio (Houston, TX, Cat No. B1036), and was dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C. CQ (chloroquinediphosphate salt, C6628) was purchased from Sigma.