Nugc4

The GC cell line SGC-7901, HGC-27, MKN45, KATOIII, BGC-823, NUGC-4 were purchased from American Type Culture Collection (ATCC,USA), and cultured in RPMI-1640 medium (HyClone, Thermo Scientific, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, Thermo Scientific, USA) and 1% penicillin/streptomycin (HyClone, Thermo Scientific, USA). HUVECs were purchased from the Sciencell and cultured in ECM consisting of 500 ml of basal medium, 25 ml of fetal bovine serum, 5 ml of endothelial cell growth supplement (ECGS) and 5 ml of penicillin/ streptomycin solution (P/S). SGC-7901 LV-YBX1 and LV-NC Cells were cultured in RPMI-1640 medium (HyClone, Thermo Scientific, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, Thermo Scientific, USA) and 1% penicillin/streptomycin (HyClone, Thermo Scientific, USA). All cells were incubated in a standard humidified incubator in 5% CO₂ at 37 °C.

Human GC cell lines, including MGC803, MKN1, HGC‐27, AGS, NUGC4, and AZ521 were purchased from the American Type Culture Collection (ATCC). All cell lines were cultured at 37 °C with 5% CO₂ in RPMI‐1640 medium (GIBCO) supplemented with 10% fetal bovine serum (FBS, GIBCO, Thermo Scientific), penicillin (100 IU/ml), and streptomycin (100 mg/mL). The USP7 inhibitor FT671 and P5091 were purchased from TargetMol (FT671, cat#1959551‐26‐8; P5091, cat# 882257‐11‐6). The investigated compounds, including DHPO, were obtained from a natural product library established in Prof. Wei‐Dong Zhang's laboratory, with purity being >95%.^[19 (link),
²⁰ (link)
^]A total of 128 pairs of fresh‐frozen GC tumor tissues and para‐tumor samples were collected from patients who underwent GC surgery in the Department of Gastric Surgery at Zhejiang Cancer Hospital (Hangzhou, China). Informed consent forms were signed in advance. Follow‐up was conducted in time to record the survival time of patients. All studies associated with clinical samples and data were carried out following the principles of the Declaration of Helsinki and were approved by the Ethics Committee of Zhejiang Cancer Hospital (study number: IRB‐2021‐456).

NUGC4, NCI-N87, SGC7901 and BGC823 were supplied by American Type Culture Collection (ATCC). We evaluated HER2 expression level in the cells by Western blot and immunohistochemistry as previously described [31 (link)]. HER2 was expressed positively in NCI-N87 and NUGC4 cells, and negatively in SGC7901 and BGC823, consistent with previous studies [13 (link), 32 (link)] (Supplementary Figure 2). The miR-21 expression in the cells showing HER2-positive expression was determined by real-time reverse-transcription polymerase chain reaction (RT-PCR) [13 (link)]. The expression of miR-21 was higher in NUGC4 SGC7901 than in NCI-N87 BGC823 cells (Supplementary Figure 3), which were therefore, selected for further study. All cells were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum and incubated at 37°C at 5% CO2 and 95% humidity.

GC cell lines (AGS, NUGC4, and MKN74) and human normal epithelial cell line (GES-1) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in the RPMI1640 medium (CD-02168-ML, GIBCO, USA) added with 10% Fetal Bovine Serum (FBS; 10270-106, GIBCO) and 100 U/mL Penicillin/Streptomycin solution in humidified incubators. The air and temperature for the cell cultivation was required of 5% CO₂ and 37 °C respectively.