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. 2023 Jul 11;26(8):107333. doi: 10.1016/j.isci.2023.107333

Figure 2.

Figure 2

PAK1 inhibition by its inhibitors or siRNA reduces the hyperpermeability of bEnd.3 endothelial monolayer in OGD model

(A) Immunoblots of p-PAK1 and PAK1 with corresponding antibodies in bEnd.3 cells at reperfusion for indicated times after 2 h OGD. GAPDH was used as a loading control.

(B and C) The statistical analysis of immunoblotted p-PAK1 (Ser144) and PAK1 proteins. Data are expressed as mean ± SEM (n = 4). ∗p < 0.05, ∗∗p < 0.01 versus control groups, one-way ANOVA with Dunnet’s post hoc test.

(D) Schematic representation of the in vitro BBB model. bEnd.3 cells were planted on microporous membrane of transwell inserts till confluence. After OGD/R treatment, the fluorescence dyes were added to upper compartment and the fluorescence intensity of lower compartment was measured 30 min later.

(E and F) The transfer rate of dextran from upper compartment to lower compartment was examined to assess the endothelial monolayer permeability. Consistent diffused 4.4 kDa TRITC-dextran or 70 kDa FITC-dextran during reoxygenation after 2 h OGD was limited by FRAX486 (1 μmol/L) or IPA-3 (10 μmol/L) treatment. Data are expressed as mean ± SEM (n = 5). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus ctrl group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus OGD + vehicle group, two-way ANOVA with Turkey’s post hoc test.

(G) The endothelial monolayer permeability was further expressed as coefficient of diffusion (in centimeters per second). FRAX486 (1 μmol/L) or IPA-3 (10 μmol/L) treatment significantly reduced the increased permeability of bEnd.3 monolayer to 4.4 kDa TRITC-dextran or 70 kDa FITC-dextran by exposure to OGD/R 3 h. Data are expressed as mean ± SEM (n = 5). ∗∗∗p < 0.001 versus ctrl group; #p < 0.05 versus OGD + vehicle (veh) group, one-way ANOVA with Sidak’s post hoc test.

(H) PAK1 expression in bEnd.3 cells is downregulated by specific siPAK1, not siNC. Data are expressed as mean ± SEM (n = 3). ∗∗∗p < 0.001, Student’s t test.

(I) Increased endothelial monolayer permeability of bEnd.3 cells to 4.4 kDa TRITC-dextran or 70 kDa FITC-dextran at OGD/R 3h was inhibited by siPAK1. Data are expressed as mean ± SEM (n = 5). ∗∗p < 0.01 versus ctrl group; #p < 0.05 versus OGD + siNC group, one-way ANOVA with Sidak’s post hoc test.

See also Figure S2.