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. Author manuscript; available in PMC: 2017 May 1.
Published in final edited form as: J Cell Physiol. 2015 Oct 19;231(5):1130–1141. doi: 10.1002/jcp.25210

Fig. 7. The Nox1/Nox4 dual inhibitor GKT137831 inhibits AT1/Nox4 binding and Ang-II-induced IL-18 and MMP9 expression.

Fig. 7

A, GKT137831 (GKT) inhibits Ang-II-induced H2O2 production. The quiescent CF were incubated with GKT (5 μM for 15 min) prior to Ang-II addition (10−7M for 30 min). H2O2 levels were quantified using the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit. B, GKT inhibits AT1/Nox4 binding. The quiescent CF were treated with GKT (5 μM for 15 min) prior to Ang-II addition (10−7 M for 15 min). AT1 and Nox4 binding was analyzed by co-IP/IB using cleared cell lysates (left and right). All blots are representatives from three independent experiments. C, GKT inhibits Ang-II-induced IL-18, IL-18Rα, and CTGF expression. The quiescent CF were treated with GKT for 15 min followed by Ang-II for 2 h. IL-18, IL-18Rα, and CTGF expression levels were analyzed by immunoblotting (n=3). D, GKT inhibits Ang-II-induced MMP-9 activation. CF made quiescent in RPMI/ITS medium were treated with GKT for 15 min followed by Ang-II for 2 h. MMP9 activation was analyzed by immunoblotting using cleared whole cell homogenates. Densitometric analysis of immunoreactive bands representing active MMP9 were semiquantified and results from three independent experiments are summarized in the bottom. E, GKT inhibits Ang-II-induced collagen expression and secretion. CF treated as in C, but for 2 h (immunoblotting) or 72 h (secretion) with Ang-II were analyzed by immunoblotting (left hand panel, n=3). Secreted collagens were analyzed by Sircol™ Collagen assay (right hand panel). F, GKT attenuates Ang-II-induced LOX activation. The quiescent CF treated as in C, but for 2 h (protein levels) or 72 h (secreted collagens) with Ang-II were analyzed by immunoblotting (left hand panel; n=3). Secreted collagens were analyzed by Sircol™ Collagen assay (right hand panel). F, GKT inhibits Ang-II-induced LOX activation. The quiescent CF were treated with GKT prior to Ang-II addition (10−7M, 24 h). LOX activity was analyzed by immunoblotting using antibodies that detect both pro and active forms (left hand panel, n=3). LOX activity was also analyzed using the conditioned medium and diaminopentane as a substrate and Amplex red as a H2O2 probe (n = 6). A, C, E, F, *P < at least 0.05 vs. untreated; †P < at least 0.05 vs. Ang-II (n=3–6).