Figure 3.

Selective inhibition of HSP90 with 17-AAG reduced the elevated sucrose movement caused by the KCl pulse across the monolayer of bEnD.3 cells. bEnd.3 cells were cultured in transwell inserts with astrocyte-conditioned media. The treatment was set as in the TEER experiments, 24 h pretreatment of 17-AAG (1 μM) or vehicle (1% DMSO in media), followed by KCl pulse (60 mM, 5 min). ¹⁴C-sucrose was added to the luminal side when KCl or aCSF was applied on the abluminal side. Samples were collected from the abluminal side after the 5 min KCl pulse (0–5 min) and 30 min after the KCl pulse (6–30 min) to detect radioactivity. (A) Setup of transwell paracellular leak model. (B) The amount of ¹⁴C-sucrose detected in the abluminal chamber was significantly increased after 5 min KCl exposure compared to aCSF controls, indicating reduced barrier integrity (KCl vs. aCSF at 5 min: ^^^ p < 0.001, as tested by one-way ANOVA with Tukey’s post-test). Treatment with the HSP90 inhibitor 17-AAG (1 μM, 24 h) prevented sucrose movement across the monolayer in KCl-treated cells, but it did not influence sucrose permeability in aCSF-treated cells (KCl + 17-AAG vs. KCl + vehicle: *** p < 0.001, assessed one-way ANOVA with Tukey’s post-test). (C) No significant difference among any groups was observed after 30 min. Values are normalized to percent of vehicle ± SEM (n = 4, dotted line).