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. 2021 Apr 29;118(3):772–784. doi: 10.1093/cvr/cvab156

Figure 6.

Figure 6

Drebrin regulates SMC ROS levels and foam cell transdifferentiation via Nox1. (A) Serial frozen sections of Dbnflox/flox/Ldlr−/− and SMC-Dbn−/−/Ldlr−/− pre-atherosclerotic aortas used in Supplementary material online, Figure S2 were incubated without (negative control) or with CellROX® Orange in the absence (total signal) or presence (nonspecific, Nox1-independent signal) of the Nox1 inhibitor ML171 (see Section 2). Fluorescence photomicrographs from single aortas of each genotype are shown; negative control samples yielded no red fluorescence (not shown). Scale bars = 50 μm. (Right), Specific CellROX® fluorescence was calculated as the fluorescence (red pixels/mm2) in aortic slices incubated with CellROX® minus that obtained from slices incubated with CellROX® plus ML171; values from five distinct aortas of each genotype were plotted, along with means ± SE. Compared with Dbnflox/flox/Ldlr−/−: *P < 0.01 (t test). (B) Frozen sections from atherosclerotic carotid interposition grafts used in Figure 3 were processed like the aortas of A; serial sections were immunostained with goat IgG specific for apoE or for no known protein (control). ‘L’, lumen. Arrows indicate the internal and external elastic laminae (and thus the tunica media). Scale bars = 50 μm. (Right) Specific CellROX® fluorescence in the tunica media was quantitated as in B. Values for four discrete carotid grafts per genotype are plotted, with means ± SE. Compared with Dbnflox/flox carotid grafts: *P < 0.03 (Mann–Whitney test). (C) SMCs from age- and sex-matched Dbn−/− and Dbnflox/flox (fl/fl) mice were solubilized and immunoblotted serially for Nox1 and β-actin. Nox1 band densities were normalized to cognate β-actin band densities; ratios were plotted as arbitrary units for four independent pairs of Dbnflox/flox and Dbn−/− SMC lines, with means ± SE. Compared with Dbnflox/flox: *P < 0.03 (Mann–Whitney test). (D) Dbn−/− and Dbnflox/flox (‘f/f’) SMCs were treated without or with the Nox1-selective inhibitor ML171 (1 μmol/L) in the presence or absence of cholesterol-methyl-β-cyclodextrin (10 µg/mL) or vehicle for 24 h and then solubilized. SMC lysates were immunoblotted sequentially for Galectin-3 and β-actin. Band densities for galectin-3 were normalized to cognate β-actin band densities; the ratios were plotted as means ± SE from three independent experiments with two independently isolated SMC lines of each genotype. Compared with cognate Dbnflox/flox SMCs: *P < 0.05 (two-way ANOVA with Sidak post hoc test).