Journal: Science Advances

Article Title: Lin28a induces SOX9 and chondrocyte reprogramming via HMGA2 and blunts cartilage loss in mice

doi: 10.1126/sciadv.abn3106

Figure Lengend Snippet: ( A ) Let-7 miRNA expression was assessed by RT-qPCR in primary chondrocytes transduced with control empty vector [Ct(EV)] or Lin28a lentivirus [Lin28a(TG)] and cultured at 1% O 2 . * P < 0.05 and *** P < 0.005 compared with empty vector control. ( B ) Heatmap representing the fold increase in the 50 most changed Let-7b and Let-7c targets in Lin28a-overexpressing chondrocytes compared to control chondrocytes in RNA-seq analysis. Red arrows indicate the genes with increase in expression ≥200%. ( C ) Let-7 target gene expression (CDC34, E2F5, HMGA2, IGFBP2, and ZBTB5) measured by RT-qPCR in primary chondrocytes transduced with [Lin28a(TG)]; * P < 0.05 compared with control empty vector [Ct(EV)]. Primary chondrocytes were treated with anti–Let-7b, anti–Let-7c, and anti–Let-7 antibody control siRNA. ( D ) Let-7 target gene expression (CDC34 and HMGA2) measured by RT-qPCR; * P < 0.05 compared with control. ( E ) Chondrocyte anabolic gene expression (Col2 and PRG4) measured by real-time qPCR; * P < 0.05 compared with controls. ( F ) Western blot analysis of MMP13 level in conditioned medium. Data are means ± SEM.

Article Snippet: Plasmids (2 μg) for Lin-28a (pMSCV- mLin28A , Addgene plasmid no. 26357) and HMGA2 (pBabe zeo HMGA2, Addgene plasmid no. 17411; pLKO.sh Hmga2 , Addgene plasmid no. 32399) or empty vector (CMV500 empty vector, Addgene plasmid no. 33348) were transfected into cells.

Techniques: Expressing, Quantitative RT-PCR, Transduction, Control, Plasmid Preparation, Cell Culture, RNA Sequencing, Targeted Gene Expression, Gene Expression, Western Blot

Journal: Science Advances

Article Title: Lin28a induces SOX9 and chondrocyte reprogramming via HMGA2 and blunts cartilage loss in mice

doi: 10.1126/sciadv.abn3106

Figure Lengend Snippet: ( A ) Western blot analysis of HMGA2 protein expression in primary chondrocytes transduced with control empty vector [Ct(EV)] or Lin28a lentivirus [Lin28a(TG)] at 1% O 2 . ( B ) Immunofluorescence staining and quantification of HMGA2 expression in cartilage from mice (scale bars, 100 μm). ( C ) Western blot analysis of HMGA2, MMP13, and SOX9 protein levels in primary chondrocytes transduced with scramble vector (shSRC), empty vector [Ct(EV)], shRNA against HMGA2 (shHMGA2), or HMGA2 lentivirus [HMGA2(TG)] at 1% O 2 . ( D ) RT-qPCR analysis of HMGA2, MMP13, and SOX9 mRNA levels in primary chondrocytes treated with shSRC or shHMGA2 at 1% O 2 . ( E ) Alcian blue staining of sulfated glycosaminoglycans (GAGs) in chondrocytes treated or not with shHMGA2 or lentivirus. Wnt3a was used to induce chondrocyte catabolism (scale bars, 200 μm). Data are means ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.005.

Article Snippet: Plasmids (2 μg) for Lin-28a (pMSCV- mLin28A , Addgene plasmid no. 26357) and HMGA2 (pBabe zeo HMGA2, Addgene plasmid no. 17411; pLKO.sh Hmga2 , Addgene plasmid no. 32399) or empty vector (CMV500 empty vector, Addgene plasmid no. 33348) were transfected into cells.

Techniques: Western Blot, Expressing, Transduction, Control, Plasmid Preparation, Immunofluorescence, Staining, shRNA, Quantitative RT-PCR

Journal: Science Advances

Article Title: Lin28a induces SOX9 and chondrocyte reprogramming via HMGA2 and blunts cartilage loss in mice

doi: 10.1126/sciadv.abn3106

Figure Lengend Snippet: ( A ) Representation of HMGA2 binding sites on SOX9 promoter. ( B ) qPCR analysis of HMGA2 binding to SOX9 regulatory regions; significance is compared with control antibody. ( C ) ChIP analysis of HMGA2 binding to the three SOX9 regulatory regions. IgG, mouse IgG (negative control). ( D ) Triple staining of Lin28a, HMGA2, and SOX9 by confocal immunofluorescence in CT and Lin28a(Tg) OA mice. Images show 10× magnification (scale bars, 500 μm) and 20× magnification (scale bars, 200 μm). ( E ) Quantification of immunofluorescence percentage of cells triple positive for Lin28a, HMGA2, and SOX9 staining. Data are means ± SEM. * P < 0.05, *** P < 0.005, and **** P < 0.001 compared with CT.

Article Snippet: Plasmids (2 μg) for Lin-28a (pMSCV- mLin28A , Addgene plasmid no. 26357) and HMGA2 (pBabe zeo HMGA2, Addgene plasmid no. 17411; pLKO.sh Hmga2 , Addgene plasmid no. 32399) or empty vector (CMV500 empty vector, Addgene plasmid no. 33348) were transfected into cells.

Techniques: Binding Assay, Control, Negative Control, Staining, Immunofluorescence

Journal: Science Advances

Article Title: Lin28a induces SOX9 and chondrocyte reprogramming via HMGA2 and blunts cartilage loss in mice

doi: 10.1126/sciadv.abn3106

Figure Lengend Snippet: ( A ) Schematic representation of the regenerative protocol applied to Col2a1-Cre ER (CT) and Lin28a flox/flox /Col2a1-Cre ER [Lin28a(Tg)] mice. OA was induced in mice at age 10 weeks, and then mice were euthanized at 4 and 8 weeks after. All mice received weekly intra-articular injection of scramble siRNA (siSCR) or siRNA against HMGA2 (si HMGA2 ). ( B ) Safranin-O staining and quantification of OA score of articular cartilage (scale bars, 100 μm). ( C ) Immunohistochemistry of MMP13 level and quantification of MMP13-positive cells (scale bars, 100 μm). ( D ) Immunohistochemistry of Ki-67 expression and quantification in mouse joints with OA (scale bars, 100 μm). ( E ) Osteophyte volume analyzed by microtomography in mice with OA. ( F ) Subchondral BV/TV analyzed by microtomography in mouse joints with OA. Data are means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005, and **** P < 0.001.

Article Snippet: Plasmids (2 μg) for Lin-28a (pMSCV- mLin28A , Addgene plasmid no. 26357) and HMGA2 (pBabe zeo HMGA2, Addgene plasmid no. 17411; pLKO.sh Hmga2 , Addgene plasmid no. 32399) or empty vector (CMV500 empty vector, Addgene plasmid no. 33348) were transfected into cells.

Techniques: Injection, Staining, Immunohistochemistry, Expressing