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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. 2. Oxidative stress contributes to Ang-II-induced CF proliferation and migration.

Fig. 2

A, The SOD mimetics attenuate Ang-II-induced CF proliferation. The quiescent CF were incubated with NAC (5mM in water for 30 min), Tempol (1 μM in DMSO), MnTBAP (100 μM in water) or MnTMPyP (1 μM in water) for 30 min prior to Ang-II (10−7M) addition. Proliferation was analyzed after 48 h. B, The antioxidant NAC and the SOD mimetics attenuate Ang-II-induced CF migration. The quiescent CF were layered on Matrigel™ basement membrane matrix-coated filters, and then treated with NAC, Tempol, MnTBAP or MnTMPyP as in A prior to Ang-II (10−7M) addition. After 12 h, cells migrating to the other side of the membrane were quantified using the MTT assay. C, The antioxidant NAC and the SOD mimetics inhibit Ang-II-induced H2O2 production and lipid peroxidation. The quiescent CF treated as in A, but for 30 min (H2O2 production) or 2 h (TBARS) with Ang-II (10−7M) were analyzed for H2O2 production using the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit. Lipid peroxidation was analyzed using the TBARS Assay kit. D, The antioxidant NAC and the SOD mimetics did not induce cell death. CF treated as in A were analyzed for mono and oligonucleosomal fragmented DNA in cytoplasmic extracts using a cell death ELISA. Total (~35 kDa) and cleaved (active form; p17/19) caspase-3 levels at 8 h were analyzed by immunoblotting using cleared whole cell lysates (n=3). A–D, * at least P < 0.01 vs. untreated, †P < at least 0.05 vs. Ang-II (n = 12).