ETX treatment causes formation of RAB7+ endosomes and MVB.
(A) Primary BEC were treated with 50nM ETX for 2 hours. Cells were stained with anti-EEA1, RAB7, or LAMP1. White arrows point to vacuolated BEC. (B) Fluorescence intensity analysis of EEA1, RAB7, and LAMP1 with and without ETX treatment. Fluorescence was normalized to untreated controls and expressed as Fold Change. Results are the mean ± STDEV. *p<0.05 determined by T-Test, n = 3–6. Changes in EEA1 (C) and RAB7 (D) staining intensity was also evaluated at indicated timepoints after 50nM ETX treatment. Fluorescence was normalized to untreated controls (0 min) and expressed as Fold Change. Results are the mean ± STDEV. *p≤0.05 determined by ANOVA versus 0 min, n = 3–6 (E) Horizontal TEM sections near the basal side in control or ETX BEC reveal ETX induced formation of numerous MVB. No increase in lysome (L) numbers was observed. Mitochondria (M) in ETX treated cells appear rounded and swollen compared to the elongated mitochondria in control treated cells.