Journal: bioRxiv

Article Title: Extracellular signalling regulates gastrin transcription through site-specific phosphorylation and nuclear redistribution of Menin

doi: 10.64898/2026.04.07.717082

Figure Lengend Snippet: (A) Schematic of FLAG-tagged C-terminal Menin deletion constructs from amino acids 470 to 610. (B) Luciferase (Luc) reporter activity in AGS, KATO III, and MKN-45G cells co-transfected with the 240 GasLuc and either empty vector, wild-type Menin, or CTD deletion constructs, normalized to Renilla Luc activity. (C) Relative mRNA expression of endogenous GAST in AGS, KATO III, and MKN-45G cells following transfection with empty vector, wild-type Menin, or deletion constructs, normalized to HPRT1 . (D) Relative MEN1 mRNA expression in AGS, KATO III, and MKN-45G cells following transfection with empty vector, wild-type Menin, or deletion constructs, normalized to GAPDH . (E) Representative Western blot analysis of total Menin protein levels in AGS, KATO III, and MKN-45G cells under basal conditions; β-tubulin served as a loading control. (F) Quantification of FLAG–Menin protein expression normalized to β-tubulin. Data are presented as mean ± SEM from three independent biological replicates. Luciferase and qRT-PCR data represent n = 3–5 independent expts, performed in triplicate. Significance was determined by one-way ANOVA with Tukey’s post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

Article Snippet: Human gastric cancer cell lines expressing unprocessed gastrin peptide, AGS (gastric adenocarcinoma; #CRL-1739) and KATO III (gastric carcinoma; #HTB-103) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and MKN-45 (gastric carcinoma; #JCRB0254) was obtained from the Japanese Collection of Research Bioresources (JCRB, Osaka, Japan).

Techniques: Construct, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Extracellular signalling regulates gastrin transcription through site-specific phosphorylation and nuclear redistribution of Menin

doi: 10.64898/2026.04.07.717082

Figure Lengend Snippet: (A, B) Representative IF images of FLAG-Menin–expressing MEF ΔMen1 and AGS cells showing FLAG-WTMenin localization (white) under the indicated treatment conditions. Arrows indicate representative cells exhibiting cytoplasmic redistribution of Menin. Scale bar, 20 μm. (D, E) Quantification of FLAG-Menin subcellular localization in MEF ΔMen1 and AGS cells. FLAG⁺ cells were scored per high-power field (HPF) and classified as nuclear (N) or cytoplasmic (C). Data are expressed as the percentage of nuclear (white) and cytoplasmic (gray/black) FLAG⁺ cells per HPF from ≥5 randomly selected fields per condition across ≥3 independent experiments. Images were analyzed at 400× Mag across the experiments. Data are presented as mean ± SEM. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple-comparison test. ***P < 0.001. (E, F) Western blot analysis of nuclear and cytoplasmic protein extracts from AGS cells transfected with wild-typw Menin, C490, or C470 constructs with or without EREG and MG132. Lamin B1 (LMNB1) and β-tubulin served as nuclear and cytoplasmic loading controls, respectively. (G, H) Western blot analysis of nuclear and cytoplasmic protein extracts from KATO III cells transfected with wild-type Menin, C490, or C470 constructs following EREG and MG132 treatments. LMNB1 and β-tubulin served as nuclear and cytoplasmic loading controls, respectively. (I) Quantification of FLAG-Menin protein expression in AGS cells normalized to loading controls. (J) Quantification of FLAG-Menin protein expression in KATO III cells normalized to loading controls. Data are presented as mean ± SEM; Two-way ANOVA with Sidak’s post hoc test. ***P < 0.01; (n = 3 biological replicates).

Article Snippet: Human gastric cancer cell lines expressing unprocessed gastrin peptide, AGS (gastric adenocarcinoma; #CRL-1739) and KATO III (gastric carcinoma; #HTB-103) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and MKN-45 (gastric carcinoma; #JCRB0254) was obtained from the Japanese Collection of Research Bioresources (JCRB, Osaka, Japan).

Techniques: Expressing, Comparison, Western Blot, Transfection, Construct

Journal: bioRxiv

Article Title: Extracellular signalling regulates gastrin transcription through site-specific phosphorylation and nuclear redistribution of Menin

doi: 10.64898/2026.04.07.717082

Figure Lengend Snippet: (A-C) Luciferase reporter assays in AGS, KATO III and MKN-45G cells expressing wild-type Menin or serial C-terminal truncation mutants (C590-C470). Cells were treated ± EREG, and luciferase activity was normalized to Renilla (Luc/Renilla, relative fold-change). (D-F) Quantitative RT-PCR analysis of endogenous GAST mRNA expression in KATO III, AGS, and MKN-45G cells expressing wild-type or truncated Menin constructs following ± EREG stimulation. Gene expression was normalized to HPRT1 and expressed as relative fold-change. (G, H) MEN1 mRNA levels in KATO III and MKN-45G cells following ± EREG treatment, normalized to GAPDH . Data are presented as mean ± SEM from n = 3-4 independent experiments performed in triplicate. Two-way ANOVA with Sidak’s post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns = not significant.

Article Snippet: Human gastric cancer cell lines expressing unprocessed gastrin peptide, AGS (gastric adenocarcinoma; #CRL-1739) and KATO III (gastric carcinoma; #HTB-103) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and MKN-45 (gastric carcinoma; #JCRB0254) was obtained from the Japanese Collection of Research Bioresources (JCRB, Osaka, Japan).

Techniques: Luciferase, Expressing, Activity Assay, Quantitative RT-PCR, Construct, Gene Expression

Journal: bioRxiv

Article Title: Extracellular signalling regulates gastrin transcription through site-specific phosphorylation and nuclear redistribution of Menin

doi: 10.64898/2026.04.07.717082

Figure Lengend Snippet: (A) Multiple sequence alignment of the Menin C-terminal region from the indicated vertebrate species showing strong conservation of a basic residue–rich motif encompassing Ser487. Conserved basic residues and Ser487 are highlighted. (B) Schematic of human Menin illustrating the position of Ser487 within NLS1. The expanded sequence highlights Ser487 and surrounding basic residues; constructs used in this study. (C) Immunoblot analysis of AGS cells expressing FLAG-tagged wild-type Menin or Ser487 mutants (S487A, S487D) following treatment with EREG, FSK, or TPA. Whole-cell lysates were probed with antibodies against phospho-Ser487 Menin, FLAG-Menin, and GAPDH. (D, E) Immunoblot analysis of MKN-45G and KATO III cells expressing wild-type Menin following stimulation with EREG, FSK, or TPA. Blots were probed for phospho-Ser487 Menin, FLAG-Menin, and β-tubulin. (F, H) Quantification of phospho-Ser487 Menin in AGS, KATO III and MKN-45G cells. (I) Immunoblot analysis of AGS cells examining activation of cAMP and EGFR downstream kinases under the indicated conditions. (J) Densitometric quantification of signalling outputs shown in (I), expressed as fold change relative to vector control. (K) Time-course of Ser487 phosphorylation in AGS cells stimulated with TPA in the presence of kinase inhibitors; MEK inhibitor (U0126), AKT inhibitor (MK-2206), PKC inhibitor (Gö6983), or combined MEK+AKT inhibition. (L) Quantification of Ser487 phosphorylation kinetics following TPA stimulation with the indicated inhibitors. (M) Area-under-the-curve (AUC) analysis of phosphorylation in (L). Data are presented as mean ± SEM; individual data points represent independent biological replicates (n = 3). Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test (*P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant).

Article Snippet: Human gastric cancer cell lines expressing unprocessed gastrin peptide, AGS (gastric adenocarcinoma; #CRL-1739) and KATO III (gastric carcinoma; #HTB-103) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and MKN-45 (gastric carcinoma; #JCRB0254) was obtained from the Japanese Collection of Research Bioresources (JCRB, Osaka, Japan).

Techniques: Sequencing, Residue, Construct, Western Blot, Expressing, Activation Assay, Plasmid Preparation, Control, Phospho-proteomics, Inhibition, Comparison

Journal: bioRxiv

Article Title: Extracellular signalling regulates gastrin transcription through site-specific phosphorylation and nuclear redistribution of Menin

doi: 10.64898/2026.04.07.717082

Figure Lengend Snippet: (A–C) Quantitative RT–PCR analysis of endogenous GAST mRNA levels in MKN-45G, AGS, and KATO III cells transfected with wild-type, S487A, or S487D Menin constructs. (D, E) Quantification of MEN1 mRNA expression following overexpression of wild-type and mutant Menin constructs in MKN-45G and AGS cells. Data represent mean ± SEM from three independent experiments. One-way ANOVA with Tukey’s multiple-comparison test (**P < 0.01; ***P < 0.001; ****P < 0.0001). (F–I) Effect of EREG or FSK treatment on endogenous GAST mRNA expression in KATO III, MKN-45G, and AGS cells expressing wild-type or mutant Menin constructs. qRT–PCR data were normalized to HPRT1 , as indicated, and expressed relative to vector control. (J, K) GAST promoter activity measured using the 240 GASLuc reporter in AGS and KATO III cells co-transfected with wild-type or mutant Menin constructs and treated with TPA. (L) AGS cells transfected with the 240 GASLuc reporter were treated with FSK in the presence or absence of kinase inhibitors following 1 h pretreatment: H89 (10 μM), MK-2206 (5 μM), U0126 (10 μM), and Gö6983 (5 μM). (M) AGS cells transfected with GASLuc were treated with TPA in the presence of the indicated inhibitors following 1 h pretreatment. Data represent mean ± SEM (n = 3 independent experiments). Statistical analysis was performed using one-way or two-way ANOVA with Tukey’s or Sidak’s multiple-comparison test, as indicated (*P < 0.05; **P < 0.01; ****P < 0.0001).

Article Snippet: Human gastric cancer cell lines expressing unprocessed gastrin peptide, AGS (gastric adenocarcinoma; #CRL-1739) and KATO III (gastric carcinoma; #HTB-103) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and MKN-45 (gastric carcinoma; #JCRB0254) was obtained from the Japanese Collection of Research Bioresources (JCRB, Osaka, Japan).

Techniques: Quantitative RT-PCR, Transfection, Construct, Expressing, Over Expression, Mutagenesis, Comparison, Plasmid Preparation, Control, Activity Assay

Journal: bioRxiv

Article Title: Extracellular signalling regulates gastrin transcription through site-specific phosphorylation and nuclear redistribution of Menin

doi: 10.64898/2026.04.07.717082

Figure Lengend Snippet: (A–C) Representative IF images and quantification of FLAG-Menin localization in MEF ΔMen1 and KATO III cells expressing wild-type Menin, phospho-null (S487A), or phospho-mimetic (S487D) constructs. Cells were treated with forskolin (FSK) to activate cAMP/PKA signaling or TPA to activate PKC signaling. FLAG-Menin is shown in white. Arrows indicate cells exhibiting cytoplasmic Menin accumulation. Scale bar, 20 μm. (B, C) Quantification of nuclear and cytoplasmic FLAG-Menin localization expressed as the percentage of FLAG-positive cells per high-power field (HPF). Data represent mean ± SEM from at least five randomly selected fields and analyzed per condition at 400× magnification. Statistical significance was determined relative to untreated controls. (D, E) Subcellular fractionation and immunoblot analysis of FLAG-Menin localization in KATO III and AGS cells expressing wild-type, S487A, or S487D constructs following pathway activation (FSK or TPA as indicated). Nuclear and cytoplasmic fractions were analyzed by immunoblot using anti-FLAG antibodies. Histone H3 and β-tubulin served as nuclear and cytoplasmic loading controls, respectively. Representative blots are shown with both high- and low-exposure FLAG-Menin signals. (F, G) Densitometric quantification of FLAG-Menin distribution between nuclear and cytoplasmic fractions in KATO III and AGS cells, normalized to the corresponding loading controls. (H, I) Quantification of nuclear and cytoplasmic Menin localization expressed as the percentage of FLAG-positive cells per HPF. Data represent mean ± SEM from at least five randomly selected fields analyzed per condition at 400× magnification. Scale bars: 20 µm. (J) IF analysis of Menin localization in AGS and MEF ΔMen1 cells under basal conditions and following treatment with TPA, the nuclear export inhibitor leptomycin B (LMB), or the proteasome inhibitor MG132, as indicated. Menin is shown in white. Data are presented as mean ± SEM from ≥3 independent experiments. Statistical analysis was performed using one-way or two-way ANOVA with Tukey’s multiple-comparison test, as indicated (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant).

Article Snippet: Human gastric cancer cell lines expressing unprocessed gastrin peptide, AGS (gastric adenocarcinoma; #CRL-1739) and KATO III (gastric carcinoma; #HTB-103) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and MKN-45 (gastric carcinoma; #JCRB0254) was obtained from the Japanese Collection of Research Bioresources (JCRB, Osaka, Japan).

Techniques: Expressing, Construct, Fractionation, Western Blot, Activation Assay, Comparison