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. 2020 Apr 6;10(12):5290–5304. doi: 10.7150/thno.42658

Figure 5.

Figure 5

PTPROt deficiency in liver macrophages aggravates inflammation and ROS production by limiting the NF-κB signaling pathway, resulting in suppressed mitophagy. A. Immunoblotting of Tomm20, Tim23, Cyto C, Atp5β, Hsp60, P62, LC3-I, LC3-II, PTPROt, and β-actin (loading control) in primary liver macrophages isolated from the mice described in Figure 1A. B. Fluorescence microscopy showing co-localization of GFP-LC3 with mitochondria [identified with the mitochondrial stain MitoTracker Deep Red (Mito-Red)] in primary liver macrophages isolated from the mice described in Figure 1A treated with CCCP for 2 hr. Bar = 20 μm. Quantification of GFP-LC3 puncta co-localized with mitochondria per cell was shown. C. Fluorescence microscopy showing co-localization of anti-Tomm20 with mitochondria [identified with the mitochondrial stain MitoTracker Deep Red (Mito-Red)] in primary liver macrophages isolated from the mice described in Figure 1A treated with CCCP for 2 hr. The arrows indicate the cells staining with MitoRed+ and Anti-Tomm20-. Bar = 100 μm. Frequency of cells with few or no mitochondria was shown. D. Immunoblotting of Tomm20, Tim23, Cyto C, Atp5β, Hsp60, P62, LC3-I, LC3-II, PTPROt, and β-actin (loading control) in RAW-Ctrl and RAW-PTPROt+ cells treated with FFAs for 24 hr. E. ELISA results showing the levels of IL-1β, TNFα, IL-6, IL-8, IL-17, and IL-18 in cell culture supernatants of RAW-Ctrl and RAW-PTPROt+ cells treated with or without free fatty acids (FFAs) and/or liensinine for 24 hr. F. Immunoblotting of NLRP3, pro-caspase-1, caspase-1-p22/p20, and β-actin (loading control) in RAW-Ctrl and RAW-PTPROt+ cells treated with CCCP and/or liensinine for 2 hr. Abbreviations: CCCP: Carbonyl cyanide m-chlorophenylhydrazone; Cyto C: Cytochrome C; DAPI: 4',6-diamidino-2-phenylindole; FFAs: Free Fatty Acids: Lien: Liensinine; MitoRed: Mitotracker Red probe; WD:Western diet.