Journal: bioRxiv

Article Title: Clinically defined mutations in MEN1 alter its tumor-suppressive function through increased menin turnover

doi: 10.1101/2022.12.20.521296

Figure Lengend Snippet: Empty vector (pcDNA), wild type menin, and menin mutant proteins bearing FLAG-epitope tags were transiently overexpressed in Men1 -null mouse embryonic fibroblasts (MEF ΔMen1 ). (A) Western blot of nuclear and cytoplasmic protein extracts probed for FLAG expression. Nuclear fractions are shown in lanes 1, 3, 5, 7, and 9 while cytoplasmic fractions are shown in lanes 2, 4, 6, 8, and 10. Histone H3 and β-tubulin were used as loading control markers for nuclear and cytoplasmic fractions, respectively. (B) Quantitation of FLAG protein band expression in nuclear and cytoplasmic compartments normalized to respective loading controls. n = 3 experimental replicates; ** = p < 0.01, *** = p < 0.001 by Two-way ANOVA with Tukey post-test; mean ± SEM. (C) Immunofluorescent images of FLAG-stained MEF ΔMen1 cells (red) co-stained with DAPI to visualize the nucleus (blue). Bottom panel shows FLAG staining in white. Scale bar = 10 μm. (D) Quantitation of FLAG expression in nuclear and cytoplasmic compartments expressed as a percentage of positively transfected cells. 10 images taken at 200X magnification were counted across n = 3 experiments for a total of 30 images per group; * = p < 0.05 by Two-way ANOVA with Tukey post-test; mean ± SEM. (E) Relative Men1 mRNA expression in menin-null MEFs following overexpression of empty vector, wild type menin, and the three mutants. n = 4 experimental replicates; n.s. by One-way ANOVA.

Article Snippet: For loading controls, membranes were re-probed with the following antibodies diluted in 5% BSA TBST for 1h at 24°C: Rabbit anti-GAPDH monoclonal antibody (1:5,000 dilution, Cat #5174, Clone D16H11, RRID: AB_10828810, Cell Signaling Technology), Rabbit anti-β-Tubulin HRP-conjugated antibody (1:2,000 dilution, Cat #5346, Clone 9F3, RRID: AB_1950376, Cell Signaling Technology), Rabbit anti-Histone H3 HRP-conjugated antibody (1:10,000 dilution, Cat #12648, Clone D1H2, RRID: AB_2797978, Cell Signaling Technology).

Techniques: Plasmid Preparation, Mutagenesis, FLAG-tag, Western Blot, Expressing, Control, Quantitation Assay, Staining, Transfection, Over Expression

Journal: bioRxiv

Article Title: Clinically defined mutations in MEN1 alter its tumor-suppressive function through increased menin turnover

doi: 10.1101/2022.12.20.521296

Figure Lengend Snippet: (A) Western blot analysis of menin and FLAG expression in whole cell extracts from AGS, MKN-45G, and GLUTag cells following overexpression of empty vector (pcDNA, lane 2), wild type menin (lane 3), and the three menin mutants (lanes 4–6). Untransfected cells (UT) were also evaluated for endogenous menin expression (lane 1). (B) Western blot analysis of FLAG expression in nuclear and cytoplasmic protein extracts in AGS, MKN-45G, and GLUTag cells. Nuclear fractions are shown in lanes 1, 3, 5, 7, and 9 while cytoplasmic fractions are shown in lanes 2, 4, 6, 8, and 10. Histone H3 and β-tubulin were used as loading control markers for nuclear and cytoplasmic fractions, respectively. (C) Quantitation of FLAG protein band intensity in nuclear and cytoplasmic compartments normalized to respective loading controls. n = 3 experimental replicates; *** = p < 0.001, **** = p < 0.0001 by Two-way ANOVA with Tukey post-test; mean ± SEM. (D) Immunofluorescent images of FLAG-stained AGS, (E) MKN-45G, and (F) GLUTag cells (red) co-stained with DAPI to visualize the nucleus (blue). Lower panels shows FLAG staining in white. Scale bar = 10 μm. (G) Quantitation of FLAG expression in nuclear and cytoplasmic compartments expressed as a percentage of positively transfected cells. 10 images taken at 200X magnification were counted across n = 3 experiments for a total of 30 images per group; * = p < 0.05 by Two-way ANOVA with Tukey post-test; mean ± SEM.

Article Snippet: For loading controls, membranes were re-probed with the following antibodies diluted in 5% BSA TBST for 1h at 24°C: Rabbit anti-GAPDH monoclonal antibody (1:5,000 dilution, Cat #5174, Clone D16H11, RRID: AB_10828810, Cell Signaling Technology), Rabbit anti-β-Tubulin HRP-conjugated antibody (1:2,000 dilution, Cat #5346, Clone 9F3, RRID: AB_1950376, Cell Signaling Technology), Rabbit anti-Histone H3 HRP-conjugated antibody (1:10,000 dilution, Cat #12648, Clone D1H2, RRID: AB_2797978, Cell Signaling Technology).

Techniques: Western Blot, Expressing, Over Expression, Plasmid Preparation, Control, Quantitation Assay, Staining, Transfection

Journal: bioRxiv

Article Title: Clinically defined mutations in MEN1 alter its tumor-suppressive function through increased menin turnover

doi: 10.1101/2022.12.20.521296

Figure Lengend Snippet: (A) Representative immunofluorescent images of vehicle-(DMSO) or MI-503 treated (1 μM) AGS cells expressing wild type menin and the three mutant proteins. Cells were immunostained for FLAG expression (red) and co-stained with the nuclear marker DAPI (blue). (B) Western blot analysis of nuclear and cytoplasmic extracts of transfected AGS cells treated with vehicle or MI-503 (1 μM). Histone H3 and β-tubulin were used as loading control markers for nuclear and cytoplasmic fractions, respectively. (C) Quantitation of FLAG protein band intensity in nuclear and cytoplasmic compartments normalized to respective loading controls. n = 4 experimental replicates; * = p < 0.05 by Two-way ANOVA with Tukey post-test; mean ± SEM. (D) Normalized optical density (OD) measurements of crystal violet dye extracted from transfected AGS cells treated with vehicle (DMSO) or MI-503 (1 μM). * = p < 0.05 by Two-way ANOVA with Tukey post-test; mean ± SEM. (E) Enzyme immunoassay measurement of plasma gastrin concentration from OMS mice before (Pre) and after (Post) treatment with vehicle, 10 mg/kg, 30 mg/kg, or 55 mg/kg of MI-503 for 4 weeks. n = 10–12 mice; ** = p < 0.01 by paired T-test; mean ± SEM. (F) Representative macroscopic images of OMS mice stomachs following treatment with vehicle or 30 and 55 mg/kg of MI-503. Arrows indicate to gastric NETs.

Article Snippet: For loading controls, membranes were re-probed with the following antibodies diluted in 5% BSA TBST for 1h at 24°C: Rabbit anti-GAPDH monoclonal antibody (1:5,000 dilution, Cat #5174, Clone D16H11, RRID: AB_10828810, Cell Signaling Technology), Rabbit anti-β-Tubulin HRP-conjugated antibody (1:2,000 dilution, Cat #5346, Clone 9F3, RRID: AB_1950376, Cell Signaling Technology), Rabbit anti-Histone H3 HRP-conjugated antibody (1:10,000 dilution, Cat #12648, Clone D1H2, RRID: AB_2797978, Cell Signaling Technology).

Techniques: Expressing, Mutagenesis, Staining, Marker, Western Blot, Transfection, Control, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Concentration Assay