Fig 1: Clinical MEN1 mutations and variants reduce the ability of menin to repress gastrin gene expression. Gastrin transcript levels (GAST) in AGS (A), MKN-45G (B), and BON-1 (C) cells following overexpression with empty vector (pcDNA), wild-type menin, and the three menin mutants. GAST mRNA was normalized to HPRT1 expression and reported as fold-change relative to pcDNA control. D, Expression of MEN1 mRNA in BON-1 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-way ANOVA with Tukey post hoc test; mean ± SEM. E, Relative GAST mRNA expression in AGS cells overexpressing menin plasmids and following serum starvation and treatment with EGF (16 hours, 40 nmol/L). F, The GasLuc system was used to evaluate human gastrin promoter activity following overexpression of empty vector (pcDNA), GasLuc plasmid alone, or GasLuc plasmid in the presence of wild-type menin and the three mutants and EGF (16 hours, 40 nmol/L). Firefly luciferase activity was normalized to the Renilla-Luciferase co-reporter. ***, P < 0.0001, by one-way ANOVA with Tukey post hoc test; mean ± SEM. Relative GAST mRNA expression in BON-1 (G) and GLUTag (H) cells overexpressing menin plasmids and following serum starvation and treatment with EGF (16 hours, 40 nmol/L). I, Immunofluorescent staining of FLAG-menin expression in GLUTag cells in the presence or absence of EGF (8 hours, 40 nmol/L) and MG132 (10 µmol/L). NT = no treatment control. J, Quantitation of FLAG-menin expression in nuclear and cytoplasmic compartments from immunofluorescence-stained images. Counts were obtained from 10 random HPF images. **, P < 0.01 = by two-way ANOVA with Tukey post hoc test; mean ± SEM. WT, wild-type.