Mkn 45

The human gastric carcinoma cell lines MKN-45, MKN7, HGC-27, and AZ521 and the normal gastric mucosal epithelial cell line GES 1 were all purchased from BNCC (BeNa Culture Collection, Beijing, China). The cell cultures were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (HyClone) in 5% CO₂ and a 37°C humidified atmosphere. The Plasmid vector pLO-ciR (GENESEED, Guangzhou, China) was used for hsa_circ_0092306 overexpression. The plasmid vector pcDNA3.1 used for PRKCB overexpression (PRKCB), miR-197-3p inhibitor, miR-197-3p mimics, and corresponding negative control (vector or NC) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The pYrbio-LT-1 plasmid (YRBIO, Changsha, China) was used to construct hsa_circ_0092306 knockdown (sh-circ) or PRKCB knockdown (sh-PRKCB) cells. The sh-RNA sequences were provided in Table S2. Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was applied to carry out cell transfection based on the protocol provided by the manufacturer.

The human gastric mucosa cell line GES-1 and the GC cell lines AGS, MKN-45, KATO III and NCI-87, procured from Bena Culture Collection, were cultured in DMEM supplemented with 10% FBS, 1% penicillin and 1% streptomycin (all from Gibco; Thermo Fisher Scientific, Inc.) at 37˚C and 5% CO₂. Cells were treated with 5 µg/ml 5-FU (Beyotime Institute of Biotechnology) at 37˚C for 48 h.

Human GC cell lines KATO III, MKN-45, SNU-1 and human normal gastric GES 1 cell line (BeNa Culture Collection); Real-time fluorescence quantitative PCR instrument (ABI 7500, Applied Biosystems, USA); Lip 2000 (TaKaRa, Dalian, China), Trizol total RNA isolation kit (TaKaRa, Dalian, China); Annexin V/PI apoptosis detection kit (TaKaRa, Dalian, China); MTT kit (Zeye Biological Technology Co., Ltd., Shanghai, China); SYBR Green qPCR Mix (TOYOBO, Japan); MTT kit (Zeye Biotechnology Co., Ltd., Shanghai, China). Electrochemiluminescence (ECL) reagent (ECL-P-100, Yanxi Biotechnology Co., Ltd., Shanghai, China), bicinchoninic acid (BCA) kit (Fermentas, Canada); Multiskan GO Full Wavelength Microplate Reader (Thermo Fisher Scientific Co., Ltd., China). The design and synthesis of all primer sequences were conducted by Sangong Bioengineering Co., Ltd., Shanghai, China (Table 1).Table 1

Primer Sequences

Gene	Forward	Reverse
miR-384	5ʹ-TGAAAATGTGGACTAGAGCCAGA’	5ʹ-CAGACACTCCAGAACAGGGC-3’
miR-134-5p	5′-CCGCTCGAGCCGGCCTTCCAACCTTTGTC-3′	5′-GAATGCGGCCGCTCCCATCATCAATATTTATTGAGCATTTAC-3′
YY1	5ʹ-GGCCGGCGTACAGTATAGATGA-3’	5ʹ-GTGCAGGGTCCGAGGT-3’
U6	5ʹ-CTCGCTTCGGCAGCACA-3’	5ʹ-AACGCTTCACGAATTTGCGT-3’
GAPDH	5ʹ-GGTCTCCTCTGACTTCAACA-3’	5ʹ-GTGAGGGTCTCTCTCTTCCT-3’

Human normal gastric epithelial cell line GES-1(RRID:CVCL_EQ22) and human GC cell lines MKN-45 (RRID:CVCL_0434), MKN-28 (RRID:CVCL_1416), HGC-27 (RRID:CVCL_1279), SNU-1 (RRID:CVCL_0099) and AGS (RRID:CVCL_0139) were purchased from the BeNa Culture Collection. GC cell line NCL-87 was purchased from the National Collection of Authenticated Cell Cultures. Cell lines were cultured in RPMI-1640 medium (HyClone; Cytiva) and maintained in a humidified atmosphere containing 5% CO₂ at 37°C. All cell cultures were supplemented with 10% fetal bovine serum (FBS; Biological Industries), 100 IU/ml penicillin and 100 µg/ml streptomycin (Gibco, Thermo Fisher Scientific, Inc.).
H. pylori clinical isolate SBK was isolated from a gastric ulcer patient and preserved in Central Lab of Weihai Municipal Hospital. CagA-knockout H. pylori SBK was established according to the previously described method (32 (link)). H. pylori strain ATCC 26695 and its corresponding CagA-knockout H. pylori (named 0547) were kindly provided by Professor Boqing Li of Binzhou Medical University (Yantai, China). All strains were maintained in Colombian agar plates supplemented with 8% sheep blood under a microaerophilic environment.

Four GC cell lines (AGS, BGC-823, MKN-45 and SGC-7901) and a gastric epithelial cell line (GES-1) were purchased from BeNa Culture Collection (Beijing China). AGS cells were cultured in Ham's F12 medium (HyClone, GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the remaining cell lines were cultured in Dulbecco's modified Eagle's medium/high glucose medium (HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS at 37°C in 5% CO₂.