Fig 2: BAP1 inactivation suppresses ASCL1 expression.A NCI-H1963 cells were treated with iBAP-II for 24 h. The protein levels of ASCL1 were determined by western blot. HSP90 was used as the internal control, n = 2. B The mRNA levels of ASCL1, GRP, DMPK, RNF183, SCN3A, MYCL, and CACNA1A were determined by real-time PCR in NCI-H1963 cells treated with either 10 or 20 μM of iBAP-II. DMSO was used as the negative control, n = 3. C The occupancy heatmaps show ASCL1 binding levels to chromatin after iBAP-II treatment. The Venn-diagram (D) and average plot (E) show the ASCL1 levels at chromatin in NCI-H1963 cells treated with either DMSO or iBAP-II. F Pathway analysis shows the most significant signaling pathways enriched in genes nearest to the loss of ASCL1 peaks (n = 11,744) induced by iBAP-II treatment. G Representative track example that shows ASCL1 occupancy between NCI-H1963 cells treated with either DMSO or iBAP-II. H NCI-H1963 cells were transduced with two distinct RING1B shRNAs. The protein levels of RING1B, H2AK119Ub, and BAP1 were determined by western blot. β-tubulin was used as the internal control, n = 2. I The mRNA levels of ASCL1 were determined by real-time PCR in NCI-H1963 cells transduced with either non-targeting gRNA or two distinct RING1B shRNAs, n = 3. J The representative tracks show the chromatin occupancy of ASXL3, BAP1, RING1B, H3K27me3, and H2AK119Ub levels at the ASCL1 gene locus in NCI-H1963 cells. K Representative tracks show the occupancy of BAP1, RING1B, H3K27me3, and H2AUb levels at chromatin. L RNA-seq was performed with NCI-H1963 cells treated with iBAP-II or transduced with non-targeting shRNA and two different RING1B gRNAs, n = 2. M The representative tracks show the expression levels of RNF2 and ASCL1 in cells transduced with either non-targeting gRNA or two distinct RING1B shRNAs. N The average plots show the levels of H3K4me1 (GSE145028), H3K27me3 (GSE164247), H2AK119Ub (upper), H3K27Ac (GSE145028), H3K4me3 (GSE164247), and Pol II (lower) at the gene body of all 739 genes that were downregulated in iBAP-II treated cells and upregulated in RING1B-depleted cells. O RNA-seq was performed with cells treated with either DMSO or EZH2 inhibitor GSK126 (2 μM) for 96 h, n = 2. The Log2FC heatmap shows the gene expression changes in the same order as L.

Fig 3: H. pylori promotes GEC apoptosis and inflammatory response via ASCL1/AQP5/Wnt/β-catenin axis.A mRNA levels of ASCL1 and AQP5 in mouse primary GECs after ASCL1 knockdown or XAV939 treatment determined by RT-qPCR. B The activation of Wnt/β-catenin pathway in mouse primary GECs after ASCL1 knockdown or XAV939 treatment detected by TOPFlash luciferase reporter assay. C mRNA levels of TNF-α and IL-1β in mouse primary GECs after ASCL1 knockdown or XAV939 treatment determined by RT-qPCR. D Protein levels of TNF-α and IL-1β in mouse primary GECs after ASCL1 knockdown or XAV939 treatment determined by ELISA. E Apoptosis of mouse primary GECs after ASCL1 knockdown or XAV939 treatment detected by Annexin V/PI staining. *p < 0.05, **p < 0.01. Data were shown as the mean ± standard deviation. Cell experiments were repeated three times independently. One-way ANOVA with Dunnett’s post hoc test was applied for the comparison of data among multiple groups.

Fig 4: H. pylori augments AQP5 expression by upregulating ASCL1.A Veen map of the transcription factors binding AQP5 predicted by hTFtarget database and AQP5-related genes in microarray data GSE106656. B, ASCL1 expression in 14 gastritis patients and 7 IM patients in microarray data GSE106656. C Correlation between ASCL1 and AQP5 expression in microarray data GSE106656. D ASCL1 mRNA level in mouse gastric mucosa measured by RT-qPCR (n = 6). E, ASCL1 mRNA level in mouse primary GECs measured by RT-qPCR. F Enrichment of ASCL1 on the AQP5 promoter detected by ChIP assay. G The silencing efficiency of si-ASCL1-1 or si-ASCL1-2 as reflected by ASCL1 mRNA level in mouse primary GECs transduced with si-ASCL1-1 or si-ASCL1-2 measured by RT-qPCR. H The target relationship between ASCL1 and AQP5 verified by dual luciferase reporter assay. I ASCL1 mRNA level in H. pylori-infected mouse primary GECs transduced with si-ASCL1 measured by RT-qPCR. *p < 0.05, **p < 0.01. Data were shown as the mean ± standard deviation. Cell experiments were repeated three times independently. Comparisons of data between two groups were analyzed by independent sample t-test. One-way ANOVA with Dunnett’s post hoc test was applied for the comparison of data among multiple groups. The data between groups at different time points were compared by repeated measures ANOVA.

Fig 5: Molecular mechanisms of H. pylori infection involved in the occurrence of gastritis.H. pylori infection induces AQP5 expression by promoting ASCL1 expression to activate the Wnt/β-catenin pathway, which led to the inflammation and apoptosis of GECs in gastritis.