Fig 1: Effects of CLIC4siRNA on development of pulmonary hypertension, Arf activation, BMPRII expression, and NF-κB activity in Sugen/hypoxia mice. (A) RVSP; (B) RV/LV+S; and (C) percentage of muscularized vessels in lungs of control mice and Sugen/hypoxia mice treated with nontargeting siRNA (control siRNA) or CLIC4 siRNA/DACC lipoplex, as indicated. D, Confocal images showing endothelial localization of nontargeting fluorescent siRNA delivered by DACC delivery vehicle (siRNACy3/DACC); Bar=10 μm. E, αSMA staining in mouse lung sections. Arrowheads point to small intrapulmonary vessels. Bar=25 μm. F–H, Graphs and corresponding representative Western blots show Arf6 and Arf1 activity, CLIC4, and BMPRII expression in lungs of the untreated and CLIC4siRNA-treated mice, as indicated. I, NF-κB activity in mice treated, as indicated. *P<0.05, **P<0.01, ***P<0.001, comparisons with controls; #P<0.05, ###P<0.001 comparisons with control siRNA Sugen/hypoxia group or as indicated. Data are presented as mean±SEM; n=7–8. One-way ANOVA with Tukey post hoc test. Arf6 indicates ADP ribosylation factor 6; BMPRII, bone morphogenetic protein receptor II; CLIC4, chloride intracellular channel 4; NF-κB, nuclear factor kappa B; LV, left ventricle; RV, right ventricular; RVSP, right ventricular systolic pressure; and VWF, von Willebrand Factor.
Fig 2: Effects of Sec7 inhibitor H3 (SecinH3) on development of pulmonary hypertension, Arf activation, BMPRII, and CLIC4 expression in lungs of MCT rats. (A) mPAP; (B) RV/LV+S; and (C) percentage of remodeled vessels in lungs of control and MCT rats treated with SecinH3, as indicated. D, Elastic van Gieson (EVG) staining showing fully muscularized peripheral arteries with double elastic laminae in MCT rat lung but single elastic laminae in control and SecinH3-treated rat lung (arrowheads). Bar=25 μm. E, Arf6 activity, (F) Arf1 activity, (G) BMPRII expression, and (H) NF-κB activity. Representative Western blots are shown below the graphs. *P<0.05, **P<0.01, ***P<0.001, comparisons with controls; #P<0.05, ##P<0.01, ###P<0.001 comparisons, as indicated. Data are presented as mean±SEM; n=6. One-way ANOVA with Tukey post hoc test. Arf6 indicates ADP ribosylation factor 6; BMPRII, bone morphogenetic protein receptor II; CLIC4, chloride intracellular channel 4; MCT, monocrotaline; and NF-κB, nuclear factor kappa B.
Fig 3: Arf6 mediates the effects of CLIC4. A, Arf6 activity; B, BMPRII expression in control HPAECs (Adcontrol), HPAECs overexpressing CLIC4 (AdCLIC4), with or without Sec7 inhibitor H3 (SecinH3; 10 mg/L, 24 h), as indicated. Representative Western blots are shown on the right. Graph in (C) and corresponding representative Western blots show the effect of control siRNA (ctrl siRNA), Arf6 siRNA, and Arf1 siRNA on BMPRII levels in Adcontrol- and AdCLIC4-overexpressing cells. D, The effect of SecinH3 on TNF-α-induced activation of NF-κB in control and CLIC4-overexpressing HPAECs; E, The effect of Arf6 siRNA and Arf1 siRNA on TNF-α-induced activation of NF-κB in cells treated, as indicated. F, Arf6 activity and CLIC4 expression in ECFCs from IPAH patients, n=6. *P<0.05, **P<0.01, ***P<0.001, comparisons with Adcontrol; #P<0.05; ##P<0.01; ###P<0.001, comparisons with AdCLIC4+TNF-α or as indicated. Data are presented as mean±SEM; n=4–6. Student t test or 1-way ANOVA with Tukey post hoc test, except for (F), where data were analyzed with Mann-Whitney U test. Arf6 indicates ADP ribosylation factor 6; BMPRII, bone morphogenetic protein receptor II; CLIC4, chloride intracellular channel 4; HPAECs, human pulmonary artery endothelial cells; NF-κB, nuclear factor kappa B; and TNF-α, tumor necrosis factor α.
Fig 4: CLIC4 increases NF-κB activity and reduces BMPRII signaling in HPAECS. A, NF-κB activity in HPAECs overexpressing CLIC4 or CLIC4shRNA in normoxia, hypoxia, and in cells treated with TNF-α (10 μg/L, 24 h) or NF-κB inhibitor, BAY 117085 (10 μmol/L, 24 h); luciferase reporter assay. (B) BMPRII protein levels, (C) Smad1/5 phosphorylation, and (D) Smad3 phosphorylation in HPAECs infected with Adcontrol or AdCLIC4. In (C), the cells were treated with BMP9 (10 μg/L, 1 h) and in (D) with TGF-β (10 μg/L, 1 h). Representative Western blots are shown underneath the graphs. E, The effect of BMPRII and PPM1A overexpression on CLIC4-induced activation of NF-κB in cells treated, as indicated. *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001, comparisons with Adcontrol; #P<0.05; ##P<0.01; ###P<0.001, comparisons with CLIC4+TNF-α or as indicated. Data are presented as mean±SEM; n=4–8. Student t test or 1-way ANOVA with Tukey’s post-test, as appropriate. BMP9 indicates bone morphogenetic protein 9; BMPRII, bone morphogenetic protein receptor II; CLIC4, chloride intracellular channel 4; HPAECs, human pulmonary artery endothelial cells; PPM1A, protein phosphatase 1A; NF-κB, nuclear factor kappa B; and TNF-α, tumor necrosis factor α.
Fig 5: Proposed CLIC4/Arf6 signaling pathway. Arf6 indicates ADP ribosylation factor 6; BMPR, bone morphogenetic protein receptor; CLIC4, chloride intracellular channel 4; GAPs, GTPase-activating proteins; GEFs, guanine exchange factors; PAH, pulmonary arterial hypertension; PPM1A, protein phosphatase 1A; NF-κB, nuclear factor kappa B; and TGF-β, transforming growth factor.
Supplier Page from Abcam for Recombinant Human CLIC4 protein (His tag N-Terminus)