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. Author manuscript; available in PMC: 2020 Dec 9.
Published in final edited form as: FEBS J. 2020 Feb 20;287(18):3967–3988. doi: 10.1111/febs.15236

Fig. 6. The engulfment (internalization) of AIEC-LF82 through epithelial TJs is impaired in ELMO1−/− EDMs with reduced recruitment of lysosomal proteins to the sites of internalization.

Fig. 6.

(A) Expression of ELMO1 protein was assessed by immunoblotting in enteroids isolated from colons of WT and ELMO1−/− mice. α-Tubulin was analyzed as a loading control. Images displayed are representative of three independent experiments (B) WT and ELMO1−/− EDMs were infected with AIEC-LF82 for 6 h followed by counting the bacterial entry using gentamicin protection assay (see Methods). Bar graphs display % internalization. Data represent the mean ± SD of three separate experiments. * indicates p≤0.05 as assayed by two-tailed Student's t-test. (C) The cellular composition of WT and ELMO1−/− enteroids were assessed by measuring the mRNA level of different cell markers; enterocyte markers (CA1 and Sucrase isomaltase), Paneth cell markers (Lysozymes and β defensin), tuft cells markers (DCLK-1), goblet cell marker (Muc-2) and stemness marker (lgr-5). The level of expression was normalized to the housekeeping gene (18s rRNA) and then the fold change was determined by comparing with the respective control (WT) as 1. Data represent the mean ± SD of three separate experiments. * indicates p≤0.05 as assayed by two-tailed Student's t-test. (D) WT and ELMO1−/− EDMs were infected with AIEC-LF82 as in B, fixed, stained with ZO1 (red), LAMP1 (green) and DAPI for nucleus, and analyzed by confocal imaging. Left: The maximum projection of Z-stacks of representative fields was shown. Insets in merged images represent magnified images and displayed at the bottom to zoom in at the point of bacterial entry through epithelial TJs. Lysosomes (marked by LAMP1) were aligned with the TJs (marked by ZO-1) in WT EDMs, but remain dispersed throughout the epithelial cell in ELMO1−/− EDMs. Lysosomes were seen in close proximity to the invading bacteria exclusively in the WT EDMs. Right: RGB plots show distance in pixels between the internalized bacteria (blue) and the TJs of host cells (red) and lysosomes (green). . Images were acquired using Confocal microscope with a Plan APO 63x objective.