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. 2021 Jun 29;7:150. doi: 10.1038/s41420-021-00548-3

Fig. 5. mTOR inhibition reversed the disturbed barrier functions induced by endothelial IRS-1 overexpression.

Fig. 5

(A) The primary endothelial cells from eTg/0 pups (PEC-eTg/0) had higher protein levels of phospho-mTOR (pmTOR), mTOR, phospho-S6K1(pS6K1), and S6K1at basal state (on P7 before HI) than the primary endothelial cells from eWT pups (PEC-eWT) (N = 6). (B, C) Representative images illustrate IgG and albumin extravasation at 24 h after HI in the cerebral cortex of eTg/0 and eWT treated with rapamycin (Rapa) or vehicle (Veh), respectively. The relative integrated optic density (IOD) of IgG and albumin signals was quantified. N = 6, Scale bar: 125um. One-Way ANOVA (Scheffe’s post-hoc test) was used for statistical analysis (b: F value = 29.083; c: F value = 4.567). All data are presented as mean ± SD. * P < 0.05, ** P < 0.001. (D) Rapamycin increased ZO-1 expression in the RECA ( + ) vessels in the eTg/0 rats compared to vehicle. Scale bar: 25um. N = 3 (E) The expression levels of pmTOR and pS6K1 were suppressed by 25 nM or 50 nM rapamycin in the IRS-1 overexpressed hCMEC clone (hCMEC-IRS-1 C1) (left panel, N = 4) and the PEC-eTg/0 (right panel, N = 4). (F) Representative images illustrate the disassembled distribution of ZO-1 in the cytoplasm induced by IRS-1 overexpression in the hCMEC-IRS-1 C1 and the PEC-eTg/0. The distribution of ZO-1 became assembled in the cell membrane by rapamycin (50 nM) treatment. Scale bar: 25 μm. N = 3.