Journal: bioRxiv

Article Title: Epigenetic Reprogramming Alters Intestinal Stem Cell Fate in Pouchitis

doi: 10.1101/2025.03.19.644201

Figure Lengend Snippet: (A-B) Western blot of c-Jun, p-c-Jun, NFκB p65, IKKα, IKKβ, p-IKKα/β, and GAPDHGAPDH in JUN knockdown (KD) versus non-targeting shRNA control (CTRL) (A). Quantification for JUN and p-JUN levels shows in (B). (C) Western blot of β-catenin and AP1 TFs following co-immunoprecipitation (Co-IP) with an antibody against c-Jun from niPB, iPB, and JUN KD niPB organoids. (D-E) Representative images (D) and quantification of generation efficiency (E) of organoid formation in shRNA control and JUN KD conditions. The green fluorescence corresponds to GFP, which is co-expressed with the shRNA to confirm successful transduction. Scale bars represent 400 μm. (F) Representative flow cytometry plots of annexin V and PI stained ISCs from shRNA control and JUN KD niPB organoids. (G) Quantification of annexin V + and annexin V + PI + ISCs from (F). (H) Immunofluorescence staining of shRNA control and JUN KD niPB organoids for CHGA, APOA1, and MUC2. Scale bars represent 50 µm. (I) Quantification of percentage of CHGA + , APOA1 + , MUC2 + , and LYZLYZ + cells in shRNA control and JUN KD niPB organoids. Dots correspond to individual organoids representing different donors and bar graphs show mean ± SEM. Statistical significance for panel C was determined using a one-tailed paired t-test, while the other panels were analyzed using a two-tailed unpaired t-test.

Article Snippet: Briefly, the c-JUN (JUN) Human shRNA Plasmid Kit (TL320397) and a scrambled shRNA control in the pGFP-C-shLenti shRNA Vector (TR30021) were purchased from Origene.

Techniques: Western Blot, Knockdown, shRNA, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, Fluorescence, Transduction, Flow Cytometry, Staining, Immunofluorescence, One-tailed Test, Two Tailed Test