Journal: bioRxiv

Article Title: Sufficient levels of BECLIN1 are required for intestinal epithelial cell homeostasis and protection against unwanted intestinal inflammation

doi: 10.1101/2025.04.29.651340

Figure Lengend Snippet: (A) Representative whole-mount immunofluorescence staining for RAB5 (red) and E-CADHERIN (green) in Becn1 +/+ , Becn1 +/- and Becn1 -/- organoids. Scale bar = 5 μm. (B) Quantification of cytoplasmic RAB5 pixels per cells. (C) Measurement of the average size of RAB5 +ve puncta. (D) Quantification of RAB5 signals on the apical and (E) lateral membranes of intestinal epithelial cells. (F) Quantification of cytoplasmic E-CADHERIN pixels per cells, along with its (G) co-localisation with cytoplasmic RAB5, as measured using Pearson’s correlation coefficient. (H) Quantification of apical and (I) lateral E-CADHERIN signals in intestinal epithelial cells. (J) Measurement of the degree of colocalisation between RAB5 and E-CADHERIN on the apical and (K) lateral membrane of intestinal epithelial cells. Data are representative of at least n = 3 different slices per organoid and of at least n = 3 biologically independent organoids. For each z-section, at least n = 3 individual IECs with clear apical to basal delineation were used. Graphs indicate the mean ± S.E.M. Statistical significance was determined using unpaired (Student’s) t-test. ns = not significant (p > 0.05).

Article Snippet: Samples were then incubated overnight at 4°C with gentle agitation using primary antibodies at the following dilutions: E-CADHERIN, 1:500 (Invitrogen, 13-1900); RAB5, 1:100 (CST, 46449); F-actin, using 1X Phalloidin-iFluor 647 solution (Abcam, ab176759).

Techniques: Immunofluorescence, Staining, Membrane