Journal: Cellular and molecular gastroenterology and hepatology
Article Title: GFAP-directed Inactivation of Men1 Exploits Glial Cell Plasticity in Favor of Neuroendocrine Reprogramming.
doi: 10.1016/j.jcmgh.2022.06.009
Figure Lengend Snippet: Figure 5. Conditional deletion of menin in GFAPD cells stimulates reprogramming from a glial-restricted progenitor lineage. (A) Whole tissue mounts of proximal duodenum from Cre-negative and tdTomato-expressing mice showing tdTomato fluorescence localized to the myenteric plexus (MP). (B) Immunofluorescent images of frozen stomach sections from WT GFAP-tdTomato mice co-stained for the glial-restricted progenitor lineage markers GFAP, S100B, and p75NTR, showing strong localization to the same cell types. Negative localization with the nerve fiber marker NF-H serves as a control. Immunofluorescent images were merged on a transmitted light micrograph to distinguish submucosal layers. Widefield images (C) and quantitation (D) of tdTomato signal in the stomach and duodenum of WT and GFAPDMen1 mice expressing tdTomato reporter. N ¼ 4–5 mice per group; ***P < .001; ****P < .0001. (E) Representative images of cryosections of corpus (CP), gastric antrum (AT), and proximal duodenum (DUO) from WT and GFAPDMen1 mice expressing tdTomato. (F) Co-immunoprecipitation (Co-IP) of menin from rat EGC lysate followed by Western blot for GFAP. Input is 5% of lysate used for IP. (G) Quantitation of band density in 3 Co-IP experiments comparing expression of GFAP with IP with IgG isotype control in cell extracts. (H) Expression of Men1 and the glial transcripts Gfap and S100b following siRNA-mediated Men1 silencing in cultured rat EGCs. N ¼ 3 independent experiments; *P < .05; ***P < .001; ****P < .0001. (I) Western blot of menin and GFAP in whole cell lysates following 72-hour Men1 silencing in rat EGCs with (J) quantitation of band density normalized to loading control. N ¼ 3 ex- periments; **P < .01; ***P < .001 by unpaired t test. All data are represented as mean ± standard deviation.
Article Snippet: A rat enteric glial cell line was purchased from ATCC (#CRL-2690) and grown in DMEM supplemented with 10% FBS and 100 U penicillin-streptomycin (EGC/PK060399egfr, ATCC, Manassas, VA).
Techniques: Expressing, Staining, Marker, Control, Quantitation Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Cell Culture, Standard Deviation
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