Fig. 7

AREG and NAMPT enhance proliferation and gene modulation in precancerous gastric cell lines and organoids. a Precancerous cell lines (Pre-EGC) and organoids were successfully established from biopsies of EGC patients and treated with AREG and/or NAMPT. b CCK-8 assay demonstrating the impact of AREG on the viability of Pre-EGC after 72 h. c qPCR analysis of Pre-EGC treated with AREG for 72 h showing changes in the expression of “initiation-promoting” genes, HER2, EGFR and PD-L1. d qPCR analysis highlighting dose-dependent modulation of PD-L1 expression in Pre-EGC following AREG treatment (with 200 ng/ml the highest). e CCK-8 assay demonstrating the impact of NAMPT (0, 25, 50, 100, 200 ng/ml) on the viability of Pre-EGC after 72 h, with significant proliferation observed at 100 ng/ml. f Dose-dependent upregulated expressions of fibroblast surface receptors ITGA5 and ITGB1, with the most significant increases observed at 25 ng/ml and 50 ng/ml, respectively. g Enhanced fluorescence intensity of CAF markers (FAP, fibronectin, α-SMA and VIM) observed in NFs co-cultured with Pre-EGC treated with 100 ng/ml NAMPT compared to normal fibroblasts (NFs) and untreated groups. h PCR analysis of Pre-EGC co-cultured with NFs treated with NAMPT for 72 h showing changes in the expression of “initiation-promoting” genes and PD-L1. i qPCR analysis highlighting dose-dependent modulation of PD-L1 expression in Pre-EGC following NAMPT treatment (significant with 100 ng/ml). j Validation and morphological characterization of precancerous gastric organoids. k ATP assay illustrating the proliferative effects of AREG on precancerous organoids after 5 days of treatment. l Organoids treated with 200 ng/ml AREG for 5 days were analyzed for size and gene expression. The left panel shows the number of organoids with diameters greater than 100 µm in the treatment group compared to controls. The right panel shows the proportion of organoids larger than 100 µm/40 µm in the treated group compared to controls. m Immunofluorescence analysis of E-cadherin in organoids treated with 200 ng/ml AREG or 100 ng/ml NAMPT for 5 days, revealing differential expression patterns. *p < 0.05, **p < 0.01, ***p < 0.01; #p < 0.05, ##p < 0.01, ###p < 0.001; ns no significance