Fig 2: Expression of GTP-RhoA, ROCK1, p-MYPT1 and p-MLC2 in the ears or lungs of mice treated with penicillin.(a) GTP-RhoA, ROCK1, p-MYPT1 and p-MLC2 expression in the ears of mice treated with 500 kU/kg penicillin for different time courses (n = 6, per group). (b) GTP-RhoA, ROCK1, p-MYPT1 and p-MLC2 expression in the lungs of mice treated with 500 kU/kg penicillin for different time courses (n = 6, per group). NS: normal saline. *p < 0.05, **p < 0.01 compared with the normal saline-treated group; one-way ANOVA. Error bars represent the s.e.m.

Fig 3: Cingulin overexpression reduces endothelial permeability in HUVECs. (A) Paracellular resistance was measured in HUVECs lentivirally transduced with cingulin–GFP vector (CGN-GFP) or GFP vector as control (GFP). CGN-overexpressing (green) and control cells (red) were stimulated with 10 µg/ml histamine, 50 ng/ml VEGF-A or 0.5 U/ml thrombin. Arrows denote the time point of stimulation. Changes in resistance at 15 min were quantified relative to the maximum response to histamine, VEGF-A or thrombin (n=3). (B) Representative western blot for the indicated proteins in CGN-overexpressing (CGN-GFP) and control cells (GFP) after histamine (10 µg/ml), VEGF-A (50 ng/ml), or thrombin (0.5 U/ml) stimulation are shown. (C) Phosphorylation of MLC2 is shown as percentage relative to that of the control (n=4). (D) Immunofluorescence staining of cingulin-overexpressing (CGN-GFP) and the control cells (GFP) for ppMLC2 (grey) and VE-cadherin (red) after 15 min of stimulation with histamine (10 µg/ml). Scale bars: 10 µm. Data are presented as the mean±s.d. *P<0.05, **P<0.01, ***P=0.001 (Student's t-test).

Fig 4: Cingulin KD increases histamine-induced permeability in HPMECs. (A) Paracellular resistance was measured in HPMECs transiently transfected with cingulin-specific siRNA (CGN KD) or nonspecific siRNA (Ctrl). CGN KD HPMECs (red) and control HPMECs (green) were stimulated with 10 µg/ml histamine. An arrow denotes the time point of stimulation of histamine. (B) Changes in resistance at 15 min were quantified relative to the maximum histamine response. (C) CGN KD was confirmed using RT-qPCR, and CGN mRNA expression is shown as fold-change relative to that of control transfected cells. (D) CGN KD was confirmed using western blotting. (E) Phosphorylation of MLC2 is shown as percentage relative to that of control HPMECs. Results demonstrate increased MLC2 phosphorylation in CGN KD cells. Data are presented as the mean±s.d. (n=3). *P<0.05, **P<0.01, ***P=0.001 (Student's t-test).

Fig 5: Loss of Cx37-/- alters the activation of downstream key signaling proteins to the Ang II signaling pathway. A through C, After a 2-hour exposure to 40 µmol/L Ang II, the levels of phosphorylated ERK (A), MLC2 (B), and AKT (C) increased in the aortas of WT but not of Cx37-/- mice. WT Ctl, WT Ang II N=3; Cx37-/- Ctl, Cx37-/- Ang II N=5. Results are means+SEM. *P=0.05 vs respective control mice and ° P=0.05, °° P=0.01 vs WT mice, as given by 2-way ANOVA. Ang II indicates angiotensin II; Ctl, control; Cx37, Connexin37; MLC2, myosin light chain 2; WT, wild type.