NLRX1 Antibody - #DF12124

Figure 1. Downregulation of NLRX1 correlates with aggravated human NP cell senescence and IDD progression. Human NP tissue specimens with different degenerative grades were collected for histological analysis. (A) Representative MRI images at T2 weight sequence were evaluated by Pfirrmann grading system. II: grade II, III: grade III, IV: grade IV. (B) histological analysis of human NP samples by alcian blue staining, scale bar: 100 μm. (C) immunohistochemical staining of CDKN2A, MKI67 and NLRX1 in different degenerative NP tissues, scale bar: 100 μm. (D and E) linear regression analyses of the tissue staining intensity of CDKN2A and that of NLRX1 (D), or the intensity of MKI67 and that of NLRX1 (E). AOD, average optical density. (F-H) protein expressions of senescence indicators (TP53, CDKN1A, CDKN2A), SASP factors (IL1B, IL6) and NLRX1 in primary human NP cells isolated from different degenerative NP tissues with the treatment of TBHP (100 μM), as determined by western blotting. (I-L) cell senescence (SA-GLB1/β-gal staining), cell proliferation (EdU incorporation) and NLRX1 expression (immunofluorescent staining) in primary human NP cells isolated from different degenerative NP tissues with the treatment of TBHP (100 μM), scale bar: 100 μm. (M and N) MRI examination, hematoxylin and eosin (HE) and safranin-O (SO) staining in sham or operation-induced degenerated disc of rat, scale bar: 500 μm (left panel), 50 μm (right panel). (O and P) immunohistochemical staining of aggrecan, collagen type II, NLRX1 and CDKN2A in sham or operation-induced degenerated disc of rat, scale bar: 500 μm (left panel), 50 μm (right panel). Data are represented as mean ± SD.

Fig. 1 Morphine induced NLRX1-mediated mitophagy in microglia. A The mitochondrial DNA (mtDNA) levels were decreased significantly in primary microglia with 1.0 μM morphine as measured by mtDNA/nDNA analysis (n = 4). B Co-staining of HSP60 or Tim23 and LC3B in primary microglia. Bar = 10 μm. C The quantified results of AD-mCherry-GFP-LC3B transfection in BV2 cells with morphine treatment (n > 50 cells). The Kruskal–Wallis test was employed to indicate statistical significance. The results of mCherry-dots are shown in red and the mCherry-GFP-dots are shown in yellow. D Representative Western blots of BV2 cells in control or morphine treatment group (n = 3–6). E The expression of NLRX1 mRNA was peaked in primary microglia with 1.0 μM morphine as measured by qPCR (n = 3). One-way ANOVA were employed in A, E. F Quantitative graphs of NLRX1 mRNA in BV2 cells (n = 6). G, H Confocal microscopy analysis of NLRX1 (red), LC3B (green) and DAPI (light blue). Bar = 2 μm. The Pearson’s correlation coefficients of NLRX1 and LC3B were elevated in morphine-treated BV2 cells (H, n = 6–9 fields). I Co-immunoprecipitation analysis of NLRX1 and LC3 in BV2 cells (n = 3). J, K Western blots analysis of NLRX1-mediated mitophagy in BV2 cells by siRNA (J, n = 5) or shRNA (K, n = 3). L The quantified results of AD-mCherry-GFP-LC3B transfection in BV2 cells with NC-siRNA or NLRX1-siRNA pre-treatment (n > 50 cells). Data represent the mean ± SEM. Student's t-test or Mann–Whitney U test were used to measure significance between two groups. (* p < 0.05, ** p < 0.01, *** p < 0.001 and ns p > 0.05)

Fig. 1 Morphine induced NLRX1-mediated mitophagy in microglia. A The mitochondrial DNA (mtDNA) levels were decreased significantly in primary microglia with 1.0 μM morphine as measured by mtDNA/nDNA analysis (n = 4). B Co-staining of HSP60 or Tim23 and LC3B in primary microglia. Bar = 10 μm. C The quantified results of AD-mCherry-GFP-LC3B transfection in BV2 cells with morphine treatment (n > 50 cells). The Kruskal–Wallis test was employed to indicate statistical significance. The results of mCherry-dots are shown in red and the mCherry-GFP-dots are shown in yellow. D Representative Western blots of BV2 cells in control or morphine treatment group (n = 3–6). E The expression of NLRX1 mRNA was peaked in primary microglia with 1.0 μM morphine as measured by qPCR (n = 3). One-way ANOVA were employed in A, E. F Quantitative graphs of NLRX1 mRNA in BV2 cells (n = 6). G, H Confocal microscopy analysis of NLRX1 (red), LC3B (green) and DAPI (light blue). Bar = 2 μm. The Pearson’s correlation coefficients of NLRX1 and LC3B were elevated in morphine-treated BV2 cells (H, n = 6–9 fields). I Co-immunoprecipitation analysis of NLRX1 and LC3 in BV2 cells (n = 3). J, K Western blots analysis of NLRX1-mediated mitophagy in BV2 cells by siRNA (J, n = 5) or shRNA (K, n = 3). L The quantified results of AD-mCherry-GFP-LC3B transfection in BV2 cells with NC-siRNA or NLRX1-siRNA pre-treatment (n > 50 cells). Data represent the mean ± SEM. Student's t-test or Mann–Whitney U test were used to measure significance between two groups. (* p < 0.05, ** p < 0.01, *** p < 0.001 and ns p > 0.05)