Journal: Cell Death Discovery

Article Title: Macrophage-derived ectosomal miR-350-3p promotes osteoarthritis progression through downregulating chondrocyte H3K36 methyltransferase NSD1

doi: 10.1038/s41420-024-01986-5

Figure Lengend Snippet: A Immunofluorescent (IF) staining of F4/80, CD206 and iNOS in synovium of sham-operated mice (CON) and DMM mice, n = 5 per group. Scale bar: 50 µm. B Quantitative PCR analysis of iNOS and ARG in Raw264.7 cells treated with 0.5 Hz, 20%-elongation strain loading for 0, 6, 12 and 24 h, n = 6 per point. C Quantitative PCR analysis of MMP13, COL2, P21 and P16 in chondrocytes co-cultured with supernatants of overloaded-RAW264.7 cells for 24 h or 48 h, n = 5 per group. D Morphology of ectosomes observed by transmission electron microscopy. Scale bar: 0.5 µm, 200 nm. E Western blot of ectosomes surface markers (performed by EchoBiotech Co. Ltd., Beijing, P. R. China). F Particle size distribution of ectosomes was measured by ZetaView nanoparticle tracking analysis. G , H Quantitative PCR and western blot of MMP13, COL2, P21 and P16 in chondrocytes treated with CON-Ectos or Tensile-Ectos for 24 h or 48 h. I Safranin O and Fast Green staining and OARSI grades of knee cartilage from DMM mice treated with CON-Ectos or Tensile-Ectos for 4 weeks, n = 5 per group. Scale bar: 100 µm. Statistical analyses were conducted by unpaired t-test ( A , I ), two-way analysis of variance followed by Sidak’s multiple comparison test ( C , G ) or one-way analysis of variance followed by Dunnett’s multiple comparison test ( B ). * P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant. OARSI Osteoarthritis Research Society International.

Article Snippet: Antibodies used for IHC/IF staining were: P21 (1:200, Abcam, ab109199), COL2 (1:200, Abclonal, A1560), MMP13 (1:800, Abclonal, A11148), F4/80 (1:100, Santa Cruz, sc-377009), SOX9 (1:500, Proteintech, 67439-1-Ig), iNOS (1:100, Santa Cruz, sc-7271), P16 INK4a (1:500, Proteintech, 10883-1-AP), NSD1 (1:100, Abclonal, A9981), CD206 (1:100, Proteintech, 18704-1-AP).

Techniques: Staining, Real-time Polymerase Chain Reaction, Cell Culture, Transmission Assay, Electron Microscopy, Western Blot, Comparison

Journal: Cell Death Discovery

Article Title: Macrophage-derived ectosomal miR-350-3p promotes osteoarthritis progression through downregulating chondrocyte H3K36 methyltransferase NSD1

doi: 10.1038/s41420-024-01986-5

Figure Lengend Snippet: A – C Quantitative PCR and western blot of COL2, MMP13, P21, P16 in chondrocytes treated with miR-350-3p mimics or mimics-NC (CON). D , I Senescence-associated β-galactosidase staining of primary chondrocytes treated with miR-350p mimics, n = 3 per group. E – G Quantitative PCR and western blot of COL2, MMP13, P21, P16 in chondrocytes treated with inhibitor-NC (CON), miR-350-3p inhibitor, CON-Ectos and Tensile-Ectos. H , J Senescence-associated β-galactosidase staining of primary chondrocytes treated with inhibitor-NC (CON), miR-350-3p inhibitor, CON-Ectos and Tensile-Ectos, n = 3 per group. Statistical analyses were performed using two-way analysis of variance followed by Sidak’s multiple comparison test ( B , C , E , G , J ) or Student’s t test ( I ). * P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant.

Article Snippet: Antibodies used for IHC/IF staining were: P21 (1:200, Abcam, ab109199), COL2 (1:200, Abclonal, A1560), MMP13 (1:800, Abclonal, A11148), F4/80 (1:100, Santa Cruz, sc-377009), SOX9 (1:500, Proteintech, 67439-1-Ig), iNOS (1:100, Santa Cruz, sc-7271), P16 INK4a (1:500, Proteintech, 10883-1-AP), NSD1 (1:100, Abclonal, A9981), CD206 (1:100, Proteintech, 18704-1-AP).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Staining, Comparison

Journal: Cell Death Discovery

Article Title: Macrophage-derived ectosomal miR-350-3p promotes osteoarthritis progression through downregulating chondrocyte H3K36 methyltransferase NSD1

doi: 10.1038/s41420-024-01986-5

Figure Lengend Snippet: A Scheme of intra-articular injection of antagomir-NC and antagomir-350-3p in DMM-OA mice. B , C , E , F Fluorescence in situ hybridization and quantification of miR-350-3p in synovium and cartilage of sham-operated mice (CON), DMM +antagomir-NC mice and DMM+antagomir-350-3p mice, n = 5 per group. Scale bar: 25 µm. D , G Safranin O and Fast Green staining and OARSI grades of the mice. Scale bar: 100 µm. H IHC/IF staining and quantification of COL2, SOX9, MMP13, P21 and P16 in cartilage from CON, DMM-OA mice treated with antagomir-NC or antagomir-350-3p. Scale bar: 50 µm. Statistical analyses were conducted using one-way analysis of variance followed by Tukey’s multiple comparison test ( E – G ). * P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant.

Article Snippet: Antibodies used for IHC/IF staining were: P21 (1:200, Abcam, ab109199), COL2 (1:200, Abclonal, A1560), MMP13 (1:800, Abclonal, A11148), F4/80 (1:100, Santa Cruz, sc-377009), SOX9 (1:500, Proteintech, 67439-1-Ig), iNOS (1:100, Santa Cruz, sc-7271), P16 INK4a (1:500, Proteintech, 10883-1-AP), NSD1 (1:100, Abclonal, A9981), CD206 (1:100, Proteintech, 18704-1-AP).

Techniques: Injection, Fluorescence, In Situ Hybridization, Staining, Immunohistochemistry-IF, Comparison

Journal: Cell Death Discovery

Article Title: Macrophage-derived ectosomal miR-350-3p promotes osteoarthritis progression through downregulating chondrocyte H3K36 methyltransferase NSD1

doi: 10.1038/s41420-024-01986-5

Figure Lengend Snippet: A KEGG pathways enrichment analysis of the differentially genes in chondrocytes overexpressed by miR-350-3p mimics. B The binding site between miR-350-3p and NSD1 predicted by ENCORI database. C – E , H Quantitative PCR and western blot of NSD1, H3K36me1, H3K36me2 in chondrocytes treated with miR-350-3p mimics or mimics-NC (CON) and miR-350-3p inhibitor or inhibitor-NC (CON). F , G IHC staining and quantification of NSD1 in cartilage. I Immunoblotting of NSD1, COL2, MMP13, P16 and P21 in chondrocytes treated with si-NSD1 or si-NC. J , K Immunoblotting of NSD1, COL2, MMP13, P16, P21, H3K36me1 and H3K36me2 in chondrocytes treated with Tensile-Ectos, si-NSD1 and miR-350-3p inhibitor. Statistical analyses were conducted using Student’s t test ( F ), two-way analysis of variance followed by Sidak’s multiple comparison test ( C , H ), one-way analysis of variance followed by Tukey’s multiple comparison test ( G ). * P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant.

Article Snippet: Antibodies used for IHC/IF staining were: P21 (1:200, Abcam, ab109199), COL2 (1:200, Abclonal, A1560), MMP13 (1:800, Abclonal, A11148), F4/80 (1:100, Santa Cruz, sc-377009), SOX9 (1:500, Proteintech, 67439-1-Ig), iNOS (1:100, Santa Cruz, sc-7271), P16 INK4a (1:500, Proteintech, 10883-1-AP), NSD1 (1:100, Abclonal, A9981), CD206 (1:100, Proteintech, 18704-1-AP).

Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Comparison

Journal: Heliyon

Article Title: Inhibition of Pard3 promotes breast cancer metastasis via the USP28 mediated deubiquitination of Snail1

doi: 10.1016/j.heliyon.2023.e22599

Figure Lengend Snippet: Pard3 knockdown stabilizes the Snail1 protein by suppressing its ubiquitination. (A) Western blot data showing that Pard3 knockdown increased Snail1 and MMP9 protein levels. In contrast, Pard3 overexpression decreased the expression of Snail1 and MMP9 proteins. (B) Western blot showing that Pard3 regulated the expression of MMP9 via Snail1. (C) RT-PCR assay showing that neither Pard3 knockdown or overexpression altered the levels of Snail1 mRNA. ns, no significance. (D) The levels of Snail1 expression after CHX (100 μg/mL) treatment were detected by Western blot. *P < 0.05, **P < 0.01, ***P < 0.001. (E) After transfection, lysates from BC cells were used for anti-Snail1 immunoprecipitation. Subsequently, western blots were performed with ubiquitin-specific antibodies. (F) Scratch assay data (Magnification, × 40) and transwell assay data (Magnification, × 200) showing that Pard3 regulated BC cell migration and invasion via Snail1. *P < 0.05, **P < 0.01.

Article Snippet: Anti -MMP9 (1:1000; A11147) antibodies was provided by ABclonal Technology Co., Ltd. (Wuhan, China).

Techniques: Knockdown, Ubiquitin Proteomics, Western Blot, Over Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Immunoprecipitation, Wound Healing Assay, Transwell Assay, Migration