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: Ablation of Lin28a in chondrocytes was induced in Lin28a tm1.2Gqda /Col2a1-Cre ER knockout homozygous (flox/flox) and heterozygous (flox/+) mice by intraperitoneal tamoxifen injection in 9-week-old mice; Col2a1-CreER (+/+) littermates were controls (CTs). OA was induced at age 10 weeks, and then mice were euthanized 8 weeks after OA induction and analyzed at age 18 weeks. ( A ) Safranin-O staining of sham and OA joints (scale bars, 100 μm). Graph represents OA score in sham and OA joints. ( B ) Immunohistochemistry of MMP13 content (scale bars, 100 μm). Graph represents the percentage of MMP13-positive cells in sham and OA mice. ( C ) Immunofluorescence of SOX9 in OA mice (scale bars, 200 μm). Graph represents the percentage of Sox9 -positive cells. ( D ) Osteophyte volume analyzed by microtomography in OA mice. ( E ) Subchondral bone volume to total volume (BV/TV) analyzed by microtomography in sham and OA mice and quantification. Data are means ± SEM. ** P < 0.01, *** P < 0.005, and **** P < 0.001.

Article Snippet: RNA-seq was performed in duplicate for the following conditions: primary murine chondrocytes were transduced for control conditions with empty vector (CMV500 empty vector; Addgene plasmid no. 33348) and for Lin28 overexpression with pMSCV-mLin28A (Addgene plasmid no. 26357).

Techniques: Knock-Out, Injection, Staining, Immunohistochemistry, 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: Wild-type mouse primary chondrocytes were transduced with Lin28a lentivirus [Lin28a(TG)] or empty vector [Ct (EV)] and cultured for 48 hours or 1 week at 1% O 2 in the presence of Wnt3a conditioned medium (Wnt3a-CM) to trigger chondrocyte catabolism. ( A ) RT-qPCR analysis of mRNA levels of stemness genes. ( B ) Western blot analysis of PRG4 and MMP13 protein expression in primary chondrocytes transduced with Ct (EV), Ct (EV), with Wnt3a-CM (wnt3a), Lin28a(TG), and Lin28a lentivirus + Wnt3a-CM [Lin28a(TG) + Wnt]. ( C ) Alcian Blue staining and spectrophotometry quantification of sulfated glycosaminoglycans in primary chondrocytes after 1 week of culture (scale bars, 200 μm). Femoral explants were harvested from 10-week-old Tg Lin28a flox/flox /Col2a1-Cre ER [Lin28a(Tg)] or Col2a1-Cre ER (CT) mice and treated with Wnt3a-CM (Wnt3a) or not (vehicle). 4-Hydroxitamoxifen was used to induce in vitro recombination. ( D ) Safranin-O staining and immunohistochemistry (collagen 2, MMP13) of CT and Lin28a(Tg) explants (scale bars, 100 μm). Graphs show the percentage of ratio of Safranin-O–unstained cartilage to Safranin-O–positive cartilage and the percentage of MMP13-positive and collagen 2–positive cells. ( E ) Apoptosis was assessed by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay and proliferation by Ki-67 immunofluorescence staining. Data are means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005, and **** P < 0.001.

Article Snippet: RNA-seq was performed in duplicate for the following conditions: primary murine chondrocytes were transduced for control conditions with empty vector (CMV500 empty vector; Addgene plasmid no. 33348) and for Lin28 overexpression with pMSCV-mLin28A (Addgene plasmid no. 26357).

Techniques: Transduction, Plasmid Preparation, Cell Culture, Quantitative RT-PCR, Western Blot, Expressing, Staining, Spectrophotometry, In Vitro, Immunohistochemistry, End Labeling, TUNEL Assay, 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 ) 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: RNA-seq was performed in duplicate for the following conditions: primary murine chondrocytes were transduced for control conditions with empty vector (CMV500 empty vector; Addgene plasmid no. 33348) and for Lin28 overexpression with pMSCV-mLin28A (Addgene plasmid no. 26357).

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: RNA-seq was performed in duplicate for the following conditions: primary murine chondrocytes were transduced for control conditions with empty vector (CMV500 empty vector; Addgene plasmid no. 33348) and for Lin28 overexpression with pMSCV-mLin28A (Addgene plasmid no. 26357).

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