primary murine chondrocytes  (Addgene inc)

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: Fig. 1. Lin28a-deficient chondrocytes exacerbate cartilage degradation in mice with OA. Ablation of Lin28a in chondrocytes was induced in Lin28atm1.2Gqda/Col2a1-CreER 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 m). Graph represents OA score in sham and OA joints. (B) Immunohistochemistry of MMP13 content (scale bars, 100 m). Graph represents the percentage of MMP13-positive cells in sham and OA mice. (C) Immunofluores- cence of SOX9 in OA mice (scale bars, 200 m). 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

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: Fig. 2. Lin28a overexpression increased extracellular matrix production in vitro. 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% O2 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 quan- tification of sulfated glycosaminoglycans in primary chondrocytes after 1 week of culture (scale bars, 200 m). Femoral explants were harvested from 10-week-old TgLin28aflox/flox/Col2a1-CreER [Lin28a(Tg)] or Col2a1-CreER (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 m). 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: Over Expression, In Vitro, Transduction, Plasmid Preparation, Cell Culture, Quantitative RT-PCR, Western Blot, Expressing, Staining, Spectrophotometry, 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: Fig. 5. Inhibition of Let-7b and Let-7c by Lin28a promoted chondrocyte reprogramming. (A) Let-7 miRNA expression was assessed by RT-qPCR in primary chondro- cytes transduced with control empty vector [Ct(EV)] or Lin28a lentivirus [Lin28a(TG)] and cultured at 1% O2. *P < 0.05 and ***P < 0.005 compared with empty vector con- trol. (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 com- pared 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: Inhibition, 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: Fig. 6. HMGA2 expression increased anabolism and reduced catabolism in chondrocytes. (A) Western blot analysis of HMGA2 protein expression in primary chon- drocytes transduced with control empty vector [Ct(EV)] or Lin28a lentivirus [Lin28a(TG)] at 1% O2. (B) Immunofluorescence staining and quantification of HMGA2 expression in cartilage from mice (scale bars, 100 m). (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% O2. (D) RT-qPCR analysis of HMGA2, MMP13, and SOX9 mRNA levels in primary chondrocytes treated with shSRC or shHMGA2 at 1% O2. (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 m). 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: Expressing, Western Blot, Transduction, Control, Plasmid Preparation, Immunofluorescence, Staining, shRNA, Quantitative RT-PCR

Journal: BMC Molecular Biology

Article Title: The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and A gene transcription

doi: 10.1186/1471-2199-7-30

Figure Lengend Snippet: Atf3 expression is upregulated during chondrocyte differentiation in vitro . Atf3 mRNA expression during chondrogenic differentiation of mouse embryonic limb bud cells in micromass culture was examined by microarray (A) and real-time PCR (B) analyses (different cell isolations were used for the experiments in A and B). Microarray data sets represent averages from three independent experiments. Real-time data were normalized to Gapdh levels and present average and SEM from four independent experiments. Both approaches show similar expression patterns with a strong increase in Atf3 expression during micromass differentiation.

Article Snippet: Micromass cultures from E11.5 mouse limb buds and isolation of primary murine chondrocytes from E15.5 long bones were performed as recently described [ , ].

Techniques: Expressing, In Vitro, Microarray, Real-time Polymerase Chain Reaction

Journal: BMC Molecular Biology

Article Title: The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and A gene transcription

doi: 10.1186/1471-2199-7-30

Figure Lengend Snippet: Atf3 expression is upregulated during chondrocyte differentiation in vivo . Tibia isolated from embryonic day 15.5. mice were microdissected into the resting/proliferating, prehypertrophic and hypertrophic areas, and RNA was isolated directly out of cartilage. Expression of type II collagen II ( Col2a1 ), type X collagen ( Col10a1 ) and Atf3 was examined by real-time PCR analyses and normalized to Gapdh expression. All data represent averages and SEM from four independent experiments (*: p < 0.05). Col2a1 was strongly expressed in the resting/proliferating area and declined subsequently (A). Col10a1 was virtually undetectable in the resting/proliferating area and strongly induced in the prehypertrophic and hypertrophic areas (B). Atf3 was already expressed in the resting/proliferating area, but expression was significantly increased in the more mature zones of the growth plate.

Article Snippet: Micromass cultures from E11.5 mouse limb buds and isolation of primary murine chondrocytes from E15.5 long bones were performed as recently described [ , ].

Techniques: Expressing, In Vivo, Isolation, Real-time Polymerase Chain Reaction

Journal: BMC Molecular Biology

Article Title: The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and A gene transcription

doi: 10.1186/1471-2199-7-30

Figure Lengend Snippet: Inhibition of actin polymerization induces Atf3 expression through transcriptional mechanisms . A) Primary chondrocytes were cultured for 24 hours with DMSO (control) or 1 μM cytochalasin D before harvest and RNA isolation. Real-time PCR demonstrated marked induction of Atf3 mRNA levels by cytochalasin D. Data represent averages and SEM from three independent experiments, normalized to Gapdh (*: p < 0.05). B) Primary chondrocytes were transfected with an Atf3 promoter plasmid and pRLCMV, followed by incubation for 24 hours with DMSO (control) or 1 μM cytochalasin D. Cells were then harvested, firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data represent averages and SEM from three independent experiments, performed in quadruplicate each (*: p < 0.05). Cytochalasin D induced Atf3 promoter activity.

Article Snippet: Micromass cultures from E11.5 mouse limb buds and isolation of primary murine chondrocytes from E15.5 long bones were performed as recently described [ , ].

Techniques: Inhibition, Expressing, Cell Culture, Control, Isolation, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Incubation, Luciferase, Activity Assay

Journal: BMC Molecular Biology

Article Title: The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and A gene transcription

doi: 10.1186/1471-2199-7-30

Figure Lengend Snippet: ATF3 suppresses Sox9-dependent transcription and stimulates Runx2-dependent transcription . Primary chondrocytes were cotransfected with SOX9 (A) or RUNX2 (B) reporter plasmids, pcDNA3 (control) or an ATF3 expression vector, and pRLCMV. 24 hours after transfection, cells were harvested, firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data represent averages and SEM from three independent experiments, performed in quadruplicate each (*: p < 0.05). ATF3 overexpression suppressed SOX9-dependent transcription and stimulated RUNX2-dependent transcription.

Article Snippet: Micromass cultures from E11.5 mouse limb buds and isolation of primary murine chondrocytes from E15.5 long bones were performed as recently described [ , ].

Techniques: Control, Expressing, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Over Expression

Journal: BMC Molecular Biology

Article Title: The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and A gene transcription

doi: 10.1186/1471-2199-7-30

Figure Lengend Snippet: ATF3 suppresses CRE-dependent transcription . Primary chondrocytes were cotransfected with a CRE reporter (A), a cyclin D1 promoter (-1745CD1LUC, B) or cyclin A promoter plasmids (p707cycAluc and p707cycAlucMut; C), pcDNA3 (control) or an ATF3 expression vector, and pRLCMV. 24 hours after transfection, cells were harvested, firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data represent averages and SEM from three independent experiments, performed in quadruplicate each (*: p < 0.05). ATF3 overexpression suppressed the CRE reporter, cyclin D1 and cyclin A wild type promoters significantly, whereas the mutant cyclin A construct showed no response to ATF3 overexpression.

Article Snippet: Micromass cultures from E11.5 mouse limb buds and isolation of primary murine chondrocytes from E15.5 long bones were performed as recently described [ , ].

Techniques: Control, Expressing, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Over Expression, Mutagenesis, Construct

Journal: BMC Molecular Biology

Article Title: The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and A gene transcription

doi: 10.1186/1471-2199-7-30

Figure Lengend Snippet: Model for ATF3 action in chondrocyte differentiation . We suggest that induction of ATF3 by differentiation stimuli antagonizes the CRE-dependent expression of cyclin D1 and cyclin A, which are induced by mitogenic stimuli through ATF2 and CREB. Reduced cyclin-dependent kinase activity and hypophosphorylation of pocket proteins in response to ATF3 upregulation then results both in cell cycle exit and in increased activity of Runx2, promoting chondrocyte differentiation.

Article Snippet: Micromass cultures from E11.5 mouse limb buds and isolation of primary murine chondrocytes from E15.5 long bones were performed as recently described [ , ].

Techniques: Expressing, Activity Assay