Fig 1: Enhanced expression of ACE in degenerated NP tissues and IL-1-induced NP cells. A-D: Representative images of HE, SO, and immunohistochemical staining for extracellular matrix components (COL2 and ACAN) and ACE demonstrated the histological assessment of human NP tissues exhibiting varying degrees of degeneration. Immunohistochemical staining with ACE antibody and western blot analysis demonstrated the expression levels of ACE protein across each group. Immunohistochemical semi-quantification of ACE (n = 3). The relative band density is measured (n = 3). Scale bar: 50 μm. E-G: Representative images of degenerative and non-degenerative NP tissues from rats, including X-ray, MRI, and histopathology (HE and SO), as well as immunofluorescence staining for ACE and extracellular matrix components (COL2 and ACAN). The average relative optical density was determined (n = 3). Scale bars: 600 μm or 100 μm. H-K: NP cells are subjected to IL-1 (10 ng/ml, 72 h). NP cells were marked with anti-ACE and degeneration markers antibodies (COL2 and ADAMTS4) as well as senescence markers antibodies (P21 and P16), exhibiting representative fluorescence pictures. The western blot analysis demonstrated the expression levels of ACE, PTEN, and phosphorylated PTEN proteins. The relative band density and mean optical density were measured (n = 3). DAPI staining of nuclei; Scale bar: 50 μm. All experiments were repeated three times. The data are presented as the mean ± SD values. One-way ANOVA and Tukey's multiple comparisons test were used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001. NP, nucleus pulposus; HE, hematoxylin and eosin; SO, safranin-o/fast green; COL2, Collagen II; ACAN, aggrecan; ADAMTS4, a disintegrin and metallo-proteinase with thrombospondin motif 4; ACE, angiotensin converting enzyme; PTEN, phosphatase and tensin homolog.
Fig 2: The effect of glycosylation regulated by ACE on ER autophagy. A and B: The Sankey and Bubble plot of metabolic pathway KEGG and GO analysis. C-D: NP cells underwent transduction with the shACE for a duration of 72 hours. OGT and O-GlcNAc were assessed via western blot analysis. Subsequently, the NP cells were marked with OGT and O-GlcNAc antibodies, exhibiting typical fluorescence pictures. The relative mean optical density was measured (n = 3). Scale bar: 50 μm. E: Forty-eight hours after shACE transfection, NP cells were treated with the TMG (10 μM) or OSMI (25 μM) thirty-six hours. Western blot analysis measured the proteins linked to degeneration (COL2 and ADAMTS4) and senescence (P21 and P16) (n = 3). F and G: NP cells transfected with OGT siRNA. P62 and LC3B were assessed via western blot analysis, and the relative band density was measured (n = 3). Subsequently, the NP cells were marked with P62 and LC3B antibodies, exhibiting typical fluorescence pictures. The relative mean optical density was measured (n = 3). Scale bar: 50 μm. H: The NP cells treated with IL-1 (10 ng/ml) were transfected with shACE. The NP cells transfected with shACE were treated with OSMI (25 μM) or not. The levels of P62 and LC3B were detected by western blot (n = 3). I: Autophagosomes/autolysosomes containing ER fragments or whorls were evaluated by transmission electron microscopy. Red arrows represent autophagosomes. Scale bars: 2 μm and 1 μm. All experiments were repeated three times. The data are presented as the mean ± SD values. One-way ANOVA and Tukey's multiple comparisons test were used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001. NP, nucleus pulposus; ACE, angiotensin converting enzyme; ER, endoplasmic reticulum; KEGG, Kyoto encyclopedia of genes and genomes; GO, gene ontology; OGT, O-linked N-acetylglucosamine transferase; O-GlcNAc, O-linked β-N-acetylglucosamine; TMG, Thiamet G; OSMI, O-GlcNAc transferase inhibitor; COL2, Collagen II; ADAMTS4, a disintegrin and metallo-proteinase with thrombospondin motif 4.
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