ETX treatment causes vacuolation in primary BEC without cell death.

(A) In vitro ETX treatment of primary BEC revealed large, perinuclear vacuole formation (white arrows) compared to untreated controls. (B) Quantification of the number of vacuolated cells in control or ETX treated BEC after two hours. Results expressed as Mean ± STDEV, *p<0.001 determined by T-Test, n = 4. (C) Cell death was evaluated in BEC after 4 hours of ETX treatment at indicated doses. Cells were treated, trypsinized, and evaluated for cell death via PI inclusion by flow cytometry. Results are expressed as the number of PI positive cells by the total number of cells (% cell death). Results expressed as Mean ± STDEV, *p<0.05 versus untreated control, determined by ANOVA, n = 3. (D) To determine if macropinocytosis, dynamin, clathrin-coated pits, or caveolae are necessary for ETX induced vacuolation, BEC were pretreated with nocadozale (Noc), Pitstop (PS), MiTMAB (MM), or filipin (Fil), respectively, for 30 minutes prior to ETX treatment. Results are expressed as the number of vacuolated cells compared to the number of vacuolated cells when treated with ETX alone (% of ETX Alone). Results expressed as mean ± STDEV of at least two separate experiments performed in triplicate. *p<0.01 versus untreated control, determined by ANOVA. (E) Live imaging of BEC cells treated with 50nM of ETX for 4 hours. PI was used to identify dead cells (red arrows). Note that vacuolated cells (white arrows) are not PI positive. (F) Quantification of the percent of cells that are PI positive only (PI+), contain vacuoles only (vacuoles), or both (PI+Vacuoles). Results expressed as mean ± STDEV, *p<0.01 determined by ANOVA, n = 5–6. (G) and (H) BEC were isolated from WT, Mal-/-, or Cav1-/- mice, and incubated with indicated ETX doses for 4 hours, and cell vacuolation or cell death were evaluated by live cell imaging, respectively. Cell death was expressed as the number of PI positive cells compared to untreated controls (% CT). Results are the mean ± STDEV. *p<0.01 determined by ANOVA versus WT, n = 5–6. (I) Evaluation of ETX oligomerization in primary BEC via western blot. BEC were treated with 50nM of ETX for indicated time points and whole cell lysates were evaluated. CHO cells expressing MAL (rMAL) treated with or without 25nM ETX for 30 minutes were used as positive and negative controls, respectively. ETX oligomers are approximately 150kDa. (J) Densitometry readings of ETX oligomers in BEC normalized to ETX oligomer detected in ETX treated CHO cells. Results are the mean ± STDEV. *p<0.05 determined by ANOVA versus 0 min, n = 2.