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. 2024 Jul 3;18(4):101376. doi: 10.1016/j.jcmgh.2024.101376

Figure 3.

Figure 3

Inhibition of H3K27me3 by EPZ6438 mitigates TRP53-induced ICC loss.(A) Activation of TRP53 by nutlin 3a (30 μmol/L, 72 hours) downregulated KIT protein, upregulated EZH2 protein, and increased H3K27me3 levels in gastric organotypic cultures from stomachs of C57BL/6 mouse aged 12–14 days old. The 150-fold less potent enantiomer nutlin 3b (30 μmol/L, 72 hours) used as control for nutlin 3a. (B) EPZ6438 (500 nmol/L) mitigated nutlin 3a-induced (30 μmol/L) upregulated H3K27me3 levels (lower left), reduced KIT protein (lower middle), and reduced SCF protein (lower right) in gastric corpus + antrum tunica muscularis organotypic cultures from 12- to 14-day-old C57BL/6J mice (n = 6/group). EPZ6438 had no effect on nutlin 3a-induce upregulated TRP53 protein (upper left), TRP53 phosphorylation (upper middle) and upregulated EZH2 protein expression (upper right). The inactive enantiomer nutlin 3b (3b; 30 μmol/L) served as a control for nutlin 3a. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Statistical significance was determined using Kruskal-Wallis 1-way analysis of variance (on ranks). (C) Reduced gastric ICC networks by nutlin 3a (30 μmol/L) in gastric corpus + antrum tunica muscularis organotypic cultures from 12- to 14-day-old C57BL/6J mice were restored by EPZ6438 (500 nmol/L) treatment. Representative confocal stacks showing KIT+ (green) and ANO1+ (magenta) myenteric ICC (ICC-MY) and intramuscular ICC (ICC-IM) in corresponding regions of the gastric corpus (greater curvature, full thickness) of nutlin 3b (3b; 30 μmol/L) + vehicle, nutlin 3a (30 μmol/L) + vehicle, and nutlin 3a (3a; 30 μmol/L) + EPZ6438 (EPZ; 500 nmol/L). n = 4 /group. Scale bar: 50 μm Statistical significance was determined using Kruskal–Wallis 1-way analysis of variance (on ranks). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. ns, not significant.