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. Author manuscript; available in PMC: 2024 Nov 15.
Published in final edited form as: J Immunol. 2023 Nov 15;211(10):1561–1577. doi: 10.4049/jimmunol.2300293

Figure 4: OxLDL accumulation in Mφs induces oxidative stress that activates Nrf2-mediated antioxidative defense.

Figure 4:

(A) The accumulation of oxLDL increases ROS in PMφs in a time-dependent manner. Representative images and quantification showing CellRox Orange staining (red) at different time points after addition of oxLDL to cultured PMφs. Data are normalized to the 0 h time point (assigned a value of 1, n=3). (B) Nrf2 (n=3) and Keap1 (n=4) protein in PMφs at different time points after addition of oxLDL. Representative immunoblots and quantification. Data are normalized and compared to the 0 h time point (assigned a value of 1). (C) OxLDL induces Nrf2-regulated gene expression in PMφs in a time-dependent manner. A heatmap shows mRNA measured by qPCR. All time points were compared to 0 h (n=6). (D) Effect of oxLDL accumulation over 24 h on Nrf2 protein in LPS-stimulated PMφs. Representative immunoblots and quantification. Values are normalized to actin and the 0 h LPS time point in cells without oxLDL (assigned a value of 1, n=4). (E) Effect of oxLDL accumulation (24 h) and LPS stimulation (6 h) on Nrf2-dependent gene expression (qPCR analysis of mRNA plotted as a heatmap). Comparisons are to PMφs without oxLDL and LPS stimulation (n=4–11). (F, G) Effect of oxLDL accumulation on Txnrd1 protein expression (F) and thioredoxin reductase (TrxR) activity (G) in LPS-stimulated PMφs. (F) Representative immunoblots and quantification normalized to actin and the 0 h LPS time point without oxLDL (assigned a value of 1, n=3). (G) Txnrd1 activity assays normalized to the group without oxLDL (assigned a value of 1, n=4, unpaired Student’s t-test). The mean ± SEM is plotted in all graphs. Unless indicated otherwise, statistical significance was determined by one-way (A-C) or two-way ANOVA (D-F), with Bonferroni correction (*P<0.05, **P<0.01, ***P<0.001).