Journal: Molecular carcinogenesis

Article Title: Breast cancer malignancy is governed by regulation of the macroH2A2/TM4SF1 axis, the AKT/NF-κB pathway, and elevated MMP13 expression.

doi: 10.1002/mc.23683

Figure Lengend Snippet: FIGURE 7 mH2A2/TM4SF1 axis regulates breast cancer cell metastatic potential via the AKT/NF‐κB signaling pathway. (A) Phosphorylation of AKT/NF‐κB was evaluated in NS or shM2 T47D cells using western blot analysis. (B) Protein expression concentration was quantified using Vilber Fusion fx software and presented in a graph. (C, D) Rescue effects of TM4SF1 on AKT/NF‐κB phosphorylation were evaluated using western blot analysis. (E) Secreted MMP13 was quantified using an enzyme‐linked immunosorbent (ELISA) assay. (F) Schematic diagram suggesting the novel role of mH2A2/TM4SF1 axis regulating breast cancer metastasis through the AKT/NF‐κB signaling pathway and MM13P activity. siC, scramble siRNA; siT, siRNA against TM4SF1. (B) p Values were calculated using one‐way ANOVA with Dunnett's multiple test for comparison between the three groups. (B, D, E) p Values were calculated using one‐way ANOVA with Dunnett's multiple test for comparison between the four groups. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: MMP13 protein levels in culture supernatants were detected using a Human MMP13 ELISA kit (KE00078, Proteintech) according to the manufacturer's instructions. https://onlinelibrary.w iley.com /doi/10.1002/m c.23683 by Indian Institute O f T echnology, W iley O nline L ibrary on [24/01/2024].

Techniques: Phospho-proteomics, Western Blot, Expressing, Concentration Assay, Software, Enzyme-linked Immunosorbent Assay, Activity Assay, Comparison

Journal: Molecular carcinogenesis

Article Title: Breast cancer malignancy is governed by regulation of the macroH2A2/TM4SF1 axis, the AKT/NF-κB pathway, and elevated MMP13 expression.

doi: 10.1002/mc.23683

Figure Lengend Snippet: FIGURE 7 mH2A2/TM4SF1 axis regulates breast cancer cell metastatic potential via the AKT/NF‐κB signaling pathway. (A) Phosphorylation of AKT/NF‐κB was evaluated in NS or shM2 T47D cells using western blot analysis. (B) Protein expression concentration was quantified using Vilber Fusion fx software and presented in a graph. (C, D) Rescue effects of TM4SF1 on AKT/NF‐κB phosphorylation were evaluated using western blot analysis. (E) Secreted MMP13 was quantified using an enzyme‐linked immunosorbent (ELISA) assay. (F) Schematic diagram suggesting the novel role of mH2A2/TM4SF1 axis regulating breast cancer metastasis through the AKT/NF‐κB signaling pathway and MM13P activity. siC, scramble siRNA; siT, siRNA against TM4SF1. (B) p Values were calculated using one‐way ANOVA with Dunnett's multiple test for comparison between the three groups. (B, D, E) p Values were calculated using one‐way ANOVA with Dunnett's multiple test for comparison between the four groups. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: MMP13 protein levels in culture supernatants were detected using a Human MMP13 ELISA kit (KE00078, Proteintech) according to the manufacturer's instructions. https://onlinelibrary.w iley.com /doi/10.1002/m c.23683 by Indian Institute O f T echnology, W iley O nline L ibrary on [24/01/2024].

Techniques: Phospho-proteomics, Western Blot, Expressing, Concentration Assay, Software, Enzyme-linked Immunosorbent Assay, Activity Assay, 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 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: Subsequently, the protein levels were measured by Fdbio-Dura ECL (Fdbio-Science) after incubation with specific secondary antibodies in the membrane for 1 h. The antibodies used in this study for western blot include: HSP70 (1:1000, Abcam, ab181606), CD81 (1:1000, Ptoteintech, 66866-1), Calnexin (1:500, Ptoteintech, 10427-2), COL2 (1:1000, Abclonal, A1560), Tsg101 (1:1000, Abcam, ab125011), MMP13 (1:1000, Abclonal, A11148), p16 INK4a (1:2000, Abcam, ab211542), NSD1 (1:500, Abclonal, A9981), Mono Methyl-Histone H3-K36 (1:2000, Abclonal, A22863), DiMethyl-Histone H3-K36 (1:10000, Abclonal, A22087), Histone H3 (1:2000, Abclonal, A17562), HRP-labeled goat IgG H&L (Jackson ImmunoResearch), GAPDH (1:8000, Proteintech, 60004-1-Ig), and P21 (1:1000, Abcam, ab109199).

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: Subsequently, the protein levels were measured by Fdbio-Dura ECL (Fdbio-Science) after incubation with specific secondary antibodies in the membrane for 1 h. The antibodies used in this study for western blot include: HSP70 (1:1000, Abcam, ab181606), CD81 (1:1000, Ptoteintech, 66866-1), Calnexin (1:500, Ptoteintech, 10427-2), COL2 (1:1000, Abclonal, A1560), Tsg101 (1:1000, Abcam, ab125011), MMP13 (1:1000, Abclonal, A11148), p16 INK4a (1:2000, Abcam, ab211542), NSD1 (1:500, Abclonal, A9981), Mono Methyl-Histone H3-K36 (1:2000, Abclonal, A22863), DiMethyl-Histone H3-K36 (1:10000, Abclonal, A22087), Histone H3 (1:2000, Abclonal, A17562), HRP-labeled goat IgG H&L (Jackson ImmunoResearch), GAPDH (1:8000, Proteintech, 60004-1-Ig), and P21 (1:1000, Abcam, ab109199).

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: Subsequently, the protein levels were measured by Fdbio-Dura ECL (Fdbio-Science) after incubation with specific secondary antibodies in the membrane for 1 h. The antibodies used in this study for western blot include: HSP70 (1:1000, Abcam, ab181606), CD81 (1:1000, Ptoteintech, 66866-1), Calnexin (1:500, Ptoteintech, 10427-2), COL2 (1:1000, Abclonal, A1560), Tsg101 (1:1000, Abcam, ab125011), MMP13 (1:1000, Abclonal, A11148), p16 INK4a (1:2000, Abcam, ab211542), NSD1 (1:500, Abclonal, A9981), Mono Methyl-Histone H3-K36 (1:2000, Abclonal, A22863), DiMethyl-Histone H3-K36 (1:10000, Abclonal, A22087), Histone H3 (1:2000, Abclonal, A17562), HRP-labeled goat IgG H&L (Jackson ImmunoResearch), GAPDH (1:8000, Proteintech, 60004-1-Ig), and P21 (1:1000, Abcam, ab109199).

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: Subsequently, the protein levels were measured by Fdbio-Dura ECL (Fdbio-Science) after incubation with specific secondary antibodies in the membrane for 1 h. The antibodies used in this study for western blot include: HSP70 (1:1000, Abcam, ab181606), CD81 (1:1000, Ptoteintech, 66866-1), Calnexin (1:500, Ptoteintech, 10427-2), COL2 (1:1000, Abclonal, A1560), Tsg101 (1:1000, Abcam, ab125011), MMP13 (1:1000, Abclonal, A11148), p16 INK4a (1:2000, Abcam, ab211542), NSD1 (1:500, Abclonal, A9981), Mono Methyl-Histone H3-K36 (1:2000, Abclonal, A22863), DiMethyl-Histone H3-K36 (1:10000, Abclonal, A22087), Histone H3 (1:2000, Abclonal, A17562), HRP-labeled goat IgG H&L (Jackson ImmunoResearch), GAPDH (1:8000, Proteintech, 60004-1-Ig), and P21 (1:1000, Abcam, ab109199).

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