Cortisol, via GR, enhances endothelial barrier integrity, and this effect is exaggerated after ZBTB16 silencing. (A) Cortisol dose-dependently increased the barrier integrity of primary CECs measured by TEER, and this was blocked by mifepristone (Mif) (n = 3 individual donors). The spectra were normalized to the TEER value 1 h before the treatment (mean ± S.E.M). (B) The fold change of cortisol effect on average TEER value across group of treatments (mean ± S.E.M). (C) Co-incubation with 1,000 nM of the selective GR antagonist, relacorilant, attenuated endothelial barrier integrity induced by 100 nM cortisol (mean ± S.D). (D) After 24 h, 100 nM cortisol (Cort) induced ZBTB16 protein expression in the nuclear fraction compared to the vehicle treatment (mean ± S.D). (E) Representative images of immunofluorescence staining of primary CECs (donor 6) that were treated with vehicle or 100 nM cortisol for 24 h. ZBTB16 (green) was present in the DAPI-stained nuclei (blue) after cortisol treatment. (F) Induction of ZBTB16 protein expression after 24 h of 1,000 nM cortisol treatment in the nuclear (Nuc) fraction was absent in ZBTB16-silenced CECs (48 h post-transfection). (G) In the ZBTB16-silenced CECs, the cortisol-induced increase in barrier integrity (normalized TEER value) was exaggerated after 24 h of treatment compared to the WT CECs (n = 2 individual donors, mean ± S.E.M). (H) The fold change of cortisol effect on average TEER value from ZBTB16-silenced CECs (mean ± S.E.M). Each experiment was performed in 3–4 technical replications. siNT: non-target control siRNA, siRNA: small interfering RNA, siZBTB16: siRNA ZBTB16, veh: vehicle.
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