Journal: Journal of Orthopaedic Translation

Article Title: Humanin reduces nucleus pulposus cells ferroptosis to alleviate intervertebral disc degeneration: An in vitro and in vivo study

doi: 10.1016/j.jot.2024.12.002

Figure Lengend Snippet: Fig. 4. HN safeguards NP cells from erastin-induced ferroptosis via increasing HSP27 expression. (A-D and F-I) Western blot analysis of PTGS2, ACSL4, GPX4, MMP13, HSP27, and Col II and quantitative assessment in various experimental groups; (E and J) Immunofluorescence staining of MMP13, Col II, GPX4, HSP27, HN, and ACSL4 and quantitative analysis across different groups (scale bar = 50 μm); (K-L) ROS detecting in NP cells and quantitative analysis in different groups(scale bar = 50 μm); (M) Quantification of GSH levels in NP cells among different experimental groups; (N) Quantification of Fe2+ levels in NP cells among different experimental groups; (O) Quantification of MDA levels in NP cells among different experimental groups; (P-Q) Calcein/PI staining of NP cells in different experi mental groups (scale bar = 200 μm). *p < 0.05, **p < 0.01, ***p < 0.001 versus CTR or NC. The values are presented as the means ± SD from at least three in dependent experiments.

Article Snippet: After antigen retrieval and blocking with 5 % normal goat serum, the slides were subjected to incubation with primary antibodies, including HSP27(1:200, 18284-1-AP, Proteintech), p-P65 (1:200, YP0191, Immunoway), GPX4(1:100, ab125066, Abcam), ACSL4 (1:200, 22401-1-AP, Proteintech), MMP13(1:100, ab39012, Abcam), Col II (1:100, ab34712, Abcam), p-STAT3(1:100, T56566, Abmart), Rattin(1:200, orb1148108, biorbyt),HN(1:1000, ABIN549044, Antibodies) and secondary antibodies.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: Journal of Orthopaedic Translation

Article Title: Humanin reduces nucleus pulposus cells ferroptosis to alleviate intervertebral disc degeneration: An in vitro and in vivo study

doi: 10.1016/j.jot.2024.12.002

Figure Lengend Snippet: Fig. 6. HN suppresses ferroptosis in NP cells and activity of NF-κB and JAK2/STAT3 pathways depend on HSP27 expression partially. (A–B) Western blot of p-JAK2, JAK2, p-STAT3, STAT3, P65, and p-P65 and quantitative analysis when HSP27 was knocked down; (C–D) Immunofluorescence staining of p-JAK2, p-STAT3, and p-P65 when HSP27 was knocked down; (E-H and J-K)Western blot of HSP27, p-JAK2,JAK2, p-STAT3, STAT3, p-P65, P65, GPX4 PTGS2, MMP13, Col II, and ACSL4 in different groups; (I and L) Immunofluorescence staining of p-STAT3, p-P65, GPX4, MMP13, Col II, and ACSL4 in different groups (scale bar = 50 or 20 μm); (M−N) ROS detecting in NP cells and quantitative analysis in different groups(scale bar = 20 μm); (O) MDA level in NP cells in different groups; (P–Q) Calcein/PI staining of NP cells in different experimental groups (scale bar = 100 μm).*p < 0.05, **p < 0.01, ***p < 0.001 versus shNC. The values are presented as the means ± SD from at least three independent experiments.

Article Snippet: After antigen retrieval and blocking with 5 % normal goat serum, the slides were subjected to incubation with primary antibodies, including HSP27(1:200, 18284-1-AP, Proteintech), p-P65 (1:200, YP0191, Immunoway), GPX4(1:100, ab125066, Abcam), ACSL4 (1:200, 22401-1-AP, Proteintech), MMP13(1:100, ab39012, Abcam), Col II (1:100, ab34712, Abcam), p-STAT3(1:100, T56566, Abmart), Rattin(1:200, orb1148108, biorbyt),HN(1:1000, ABIN549044, Antibodies) and secondary antibodies.

Techniques: Activity Assay, Expressing, Western Blot, Immunofluorescence, Staining

Journal: Journal of Orthopaedic Translation

Article Title: Humanin reduces nucleus pulposus cells ferroptosis to alleviate intervertebral disc degeneration: An in vitro and in vivo study

doi: 10.1016/j.jot.2024.12.002

Figure Lengend Snippet: Fig. 8. HN alleviates IDD by inhibiting NP cells ferroptosis in rats. (A) The experimental procedure conducted in vivo; (B) ELISA for detecting FITC-HN In rat serum at the indicated time points; (C) Within 30 min after intranasal treatment, FITC-HN was detected associated with the IVD; (D–E) MRI images, H&E, and Safranin-O/fast green staining of rat discs in different groups (scale bar = 100 μm); (F) Histopathological score were utilized to assess the extent of IDD in different groups; (G–H) IHC staining of p-STAT3, p-P65, HSP27,and Rattin and quantitative analysis in different groups(scale bar = 100 μm); (I) Immunofluorescence of GPX4, ACSL4, Col II, and MMP13 in different groups (scale bar = 100 μm). *p < 0.05, **p < 0.01, ***p < 0.001 versus Sham or NS + IDD. Values are presented as the mean ± SD from at least three independent experiments.

Article Snippet: After antigen retrieval and blocking with 5 % normal goat serum, the slides were subjected to incubation with primary antibodies, including HSP27(1:200, 18284-1-AP, Proteintech), p-P65 (1:200, YP0191, Immunoway), GPX4(1:100, ab125066, Abcam), ACSL4 (1:200, 22401-1-AP, Proteintech), MMP13(1:100, ab39012, Abcam), Col II (1:100, ab34712, Abcam), p-STAT3(1:100, T56566, Abmart), Rattin(1:200, orb1148108, biorbyt),HN(1:1000, ABIN549044, Antibodies) and secondary antibodies.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Immunofluorescence

Journal: International journal of biological macromolecules

Article Title: Aberrant serum-derived FN1 variants bind to integrin β1 on glomerular endothelial cells contributing to thin basement membrane nephropathy.

doi: 10.1016/j.ijbiomac.2024.136282

Figure Lengend Snippet: Fig. 5. Expression changes of key GBM proteins and their response to FN1 Variants in TBMN. Immunofluorescence experiments assessed the expression of Laminin α5β2, COL4A3/4/5 and Integrin β1 in the GBM. The findings revealed non-homogeneous linear structure expression of these key GBM proteins in the presence of FN1 variants, indicating their potential involvement in the pathogenesis of TBMN. HC = healthy control.

Article Snippet: Incubating primary antibodies overnight at 4 ◦C, with specific primary antibodies against HA (1:2000, ab9110, Abcam), Integrin β1 (1:1000, 12,594–1-AP, Proteintech), β-tubulin (1:3000, ab0039, Abways), Laminin α5 (1:1000, ab210957, Abcam), Laminin β2 (1:1000, ab210956, Abcam), Laminin γ1 (1:1000, ab233389, Abcam) in Tris-Buffered Saline Tween-20 (TBST) containing 5 % skim milk.

Techniques: Expressing, Immunofluorescence, Control

Journal: International journal of biological macromolecules

Article Title: Aberrant serum-derived FN1 variants bind to integrin β1 on glomerular endothelial cells contributing to thin basement membrane nephropathy.

doi: 10.1016/j.ijbiomac.2024.136282

Figure Lengend Snippet: Fig. 6. Abnormal co-localization of FN1 variants with Integrin β1 and its impact on other GBM components. Immunofluorescence studies investigated the rela tionship between FN1 variants and the expression patterns of GBM components. The findings reveal abnormal co-localization of FN1 variants with Integrin β1, accompanied by reduced co-localization of other GBM components. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Incubating primary antibodies overnight at 4 ◦C, with specific primary antibodies against HA (1:2000, ab9110, Abcam), Integrin β1 (1:1000, 12,594–1-AP, Proteintech), β-tubulin (1:3000, ab0039, Abways), Laminin α5 (1:1000, ab210957, Abcam), Laminin β2 (1:1000, ab210956, Abcam), Laminin γ1 (1:1000, ab233389, Abcam) in Tris-Buffered Saline Tween-20 (TBST) containing 5 % skim milk.

Techniques: Immunofluorescence, Expressing

Journal: International journal of biological macromolecules

Article Title: Aberrant serum-derived FN1 variants bind to integrin β1 on glomerular endothelial cells contributing to thin basement membrane nephropathy.

doi: 10.1016/j.ijbiomac.2024.136282

Figure Lengend Snippet: Fig. 7. Competitive Binding of FN1 Variants to Integrin β1. (A and B) Duolink proximity ligation assay and Co-IP were employed to analyze the protein interaction between FN1 variants and Integrin β1. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Incubating primary antibodies overnight at 4 ◦C, with specific primary antibodies against HA (1:2000, ab9110, Abcam), Integrin β1 (1:1000, 12,594–1-AP, Proteintech), β-tubulin (1:3000, ab0039, Abways), Laminin α5 (1:1000, ab210957, Abcam), Laminin β2 (1:1000, ab210956, Abcam), Laminin γ1 (1:1000, ab233389, Abcam) in Tris-Buffered Saline Tween-20 (TBST) containing 5 % skim milk.

Techniques: Binding Assay, Proximity Ligation Assay, Co-Immunoprecipitation Assay