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. 2019 Nov 11;2019:1278301. doi: 10.1155/2019/1278301

Figure 5.

Figure 5

PDBu-stimulated hydrogen peroxide generation in activated human macrophages. PDBu-differentiated THP-1 macrophages were left untreated (MΦ) or activated with IFN-γ+LPS or IL-4 for 72 hours. (a) Average trace depicting the accumulation of PDBu (10 μM)-stimulated hydrogen peroxide in the culture media over 90 min detected via Amplex Red fluorescence. (b) Hydrogen peroxide concentration was calculated at 90 minutes and expressed as mean concentration ± SEM. (c) M(IFN-γ+LPS) and M(IL-4) hydrogen peroxide concentration expressed as a fold change relative to the untreated macrophage (MΦ) control, n = 8. (d) Representative histogram and gating strategy for DCF+ macrophages, depicting M1 versus M2 polarisation and the effect of catalase-polyethylene glycol (PEG-Cat; 1000 U/ml) on the M2 signal. (e) Mean DCF+ macrophages, normalised to the response in untreated macrophages (MΦ), n = 7. (f) SOD1, (g) SOD2, and (h) SOD3 mRNA expression in activated THP-1 macrophages at 24, 48, and 72 hours, determined by RT-qPCR and expressed relative to the average MΦ (untreated) value, n = 5‐8. (i) SOD2 and (j) SOD3 protein expression in activated THP-1 macrophages at 72 hours, determined by western blotting, n = 6. Representative blots, depicting for n = 3, shown on RHS, with GAPDH used as a loading control. All results expressed as mean ± SEM, ∗∗P < 0.01, ∗∗∗P < 0.001 (1-way ANOVA followed by Dunnett's post hoc test (g, h, i) or Student's unpaired t-test (c, e)).