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    ATCC mouse pluripotent mesenchymal precursor cell line c2c12
    Involvement of Smads in the induction of Runx2 expression. (A) Control <t>C2C12</t> cells and C2C12-Sm5 cells were treated with the indicated concentration of BMP-2 for 6 h. Total RNA was prepared from the cells, and the Runx2 mRNA level was analyzed by Northern blotting using Rx2-Ct as a probe. (B) Protein lysates were obtained from control C2C12 (lane 1) and C2C12-Sm5 (lane 2) cells, and the level of Runx2 protein was analyzed by Western blotting. (C) C2C12 cells were cultured in the absence or presence of BMP-2 (300 ng/ml) and cycloheximide (CHX; 5 μg/ml) as indicated for 6 h. Total RNAs were prepared, and the Runx2 mRNA level was analyzed by Northern blot hybridization using the Rx2-Ct probe.
    Mouse Pluripotent Mesenchymal Precursor Cell Line C2c12, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Involvement of Smads in the induction of Runx2 expression. (A) Control C2C12 cells and C2C12-Sm5 cells were treated with the indicated concentration of BMP-2 for 6 h. Total RNA was prepared from the cells, and the Runx2 mRNA level was analyzed by Northern blotting using Rx2-Ct as a probe. (B) Protein lysates were obtained from control C2C12 (lane 1) and C2C12-Sm5 (lane 2) cells, and the level of Runx2 protein was analyzed by Western blotting. (C) C2C12 cells were cultured in the absence or presence of BMP-2 (300 ng/ml) and cycloheximide (CHX; 5 μg/ml) as indicated for 6 h. Total RNAs were prepared, and the Runx2 mRNA level was analyzed by Northern blot hybridization using the Rx2-Ct probe.

    Journal: Molecular and Cellular Biology

    Article Title: Runx2 Is a Common Target of Transforming Growth Factor ?1 and Bone Morphogenetic Protein 2, and Cooperation between Runx2 and Smad5 Induces Osteoblast-Specific Gene Expression in the Pluripotent Mesenchymal Precursor Cell Line C2C12

    doi:

    Figure Lengend Snippet: Involvement of Smads in the induction of Runx2 expression. (A) Control C2C12 cells and C2C12-Sm5 cells were treated with the indicated concentration of BMP-2 for 6 h. Total RNA was prepared from the cells, and the Runx2 mRNA level was analyzed by Northern blotting using Rx2-Ct as a probe. (B) Protein lysates were obtained from control C2C12 (lane 1) and C2C12-Sm5 (lane 2) cells, and the level of Runx2 protein was analyzed by Western blotting. (C) C2C12 cells were cultured in the absence or presence of BMP-2 (300 ng/ml) and cycloheximide (CHX; 5 μg/ml) as indicated for 6 h. Total RNAs were prepared, and the Runx2 mRNA level was analyzed by Northern blot hybridization using the Rx2-Ct probe.

    Article Snippet: The mouse pluripotent mesenchymal precursor cell line C2C12 was purchased from the American Type Culture Collection C2C12 and MC3T3-E1 cells were maintained in Dulbecco modified Eagle medium (DMEM) containing 15% fetal bovine serum (FBS), and penicillin G (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere of 5% CO2 in air.

    Techniques: Expressing, Concentration Assay, Northern Blot, Western Blot, Cell Culture, Hybridization

    A model for the common and distinct activities of TGF-β and BMP in C2C12 cells. Runx2 is an indirect target of the Smad signaling pathway. Induction of Runx2 is essential for the common activities of TGF-β and BMP. Ligand-specific gene expression, however, requires cooperation between Runx2 and receptor-activated Smads (R-Smad).

    Journal: Molecular and Cellular Biology

    Article Title: Runx2 Is a Common Target of Transforming Growth Factor ?1 and Bone Morphogenetic Protein 2, and Cooperation between Runx2 and Smad5 Induces Osteoblast-Specific Gene Expression in the Pluripotent Mesenchymal Precursor Cell Line C2C12

    doi:

    Figure Lengend Snippet: A model for the common and distinct activities of TGF-β and BMP in C2C12 cells. Runx2 is an indirect target of the Smad signaling pathway. Induction of Runx2 is essential for the common activities of TGF-β and BMP. Ligand-specific gene expression, however, requires cooperation between Runx2 and receptor-activated Smads (R-Smad).

    Article Snippet: The mouse pluripotent mesenchymal precursor cell line C2C12 was purchased from the American Type Culture Collection C2C12 and MC3T3-E1 cells were maintained in Dulbecco modified Eagle medium (DMEM) containing 15% fetal bovine serum (FBS), and penicillin G (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere of 5% CO2 in air.

    Techniques: Expressing

    Suppression of Runx2 expression during myogenic differentiation. C2C12 cells were cultured in DMEM containing 5% FBS for 1 day (lane 1) or 7 days (lane 2). Total RNA was prepared, and the levels of myoD and Runx2 mRNA were analyzed by Northern blotting.

    Journal: Molecular and Cellular Biology

    Article Title: Runx2 Is a Common Target of Transforming Growth Factor ?1 and Bone Morphogenetic Protein 2, and Cooperation between Runx2 and Smad5 Induces Osteoblast-Specific Gene Expression in the Pluripotent Mesenchymal Precursor Cell Line C2C12

    doi:

    Figure Lengend Snippet: Suppression of Runx2 expression during myogenic differentiation. C2C12 cells were cultured in DMEM containing 5% FBS for 1 day (lane 1) or 7 days (lane 2). Total RNA was prepared, and the levels of myoD and Runx2 mRNA were analyzed by Northern blotting.

    Article Snippet: The mouse pluripotent mesenchymal precursor cell line C2C12 was purchased from the American Type Culture Collection C2C12 and MC3T3-E1 cells were maintained in Dulbecco modified Eagle medium (DMEM) containing 15% fetal bovine serum (FBS), and penicillin G (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere of 5% CO2 in air.

    Techniques: Expressing, Cell Culture, Northern Blot

    ) and three mutant DNAs, M1 (one mismatch in the putative Runx binding site [CACCACA]), M2 (a perfect match [GACCACA]), and M3 (putative Smad binding site [CAGACA]). Putative Runx2 and Smad binding sites are indicated by solid and dotted underlining, respectively, and mutated nucleotides are marked by asterisks. (B) EMSA was performed by using the TβRE probe and nuclear lysates obtained from C2C12 cells untreated or treated with TGF-β1 or BMP-2 for 24 h. The reactions were performed in the presence or absence of PEBP2β/Cbfβ protein. A 50-fold molar excess of unlabeled mutant oligonucleotide was incubated with the nuclear lysates as competitor DNA (Ct). The arrow and arrowhead indicate the TβRE binding complex and free probe, respectively. (C) EMSA was performed by using the same nuclear lysates and TβRE probes in the presence or absence of a polyclonal antibody which recognized all members of the Runx α subunit (anti-α; lane 2 and 5) or β subunit (anti-β; lane 3 and 6) or a monoclonal antibody which specifically recognized Runx2 (anti-Runx2; lanes 8, 10, and 12). The arrowheads and arrows indicate the positions of Runx2 and Runx-antibody complexes, respectively. (D) Time course induction of Runx2 was analyzed by EMSA using nuclear lysates prepared from cells treated with TGF-β1 for 0, 4, 12, 18, 24, 72, and 120 h. A nuclear extract prepared from cells cultured for 72 h in the absence of TGF-β1 was used as a control. EMSA was performed with the TβRE probe in the presence or absence of a 50-fold molar excess of unlabeled M3 as competitor. The arrow indicates the TβRE binding complex. Ct, competitor.

    Journal: Molecular and Cellular Biology

    Article Title: Runx2 Is a Common Target of Transforming Growth Factor ?1 and Bone Morphogenetic Protein 2, and Cooperation between Runx2 and Smad5 Induces Osteoblast-Specific Gene Expression in the Pluripotent Mesenchymal Precursor Cell Line C2C12

    doi:

    Figure Lengend Snippet: ) and three mutant DNAs, M1 (one mismatch in the putative Runx binding site [CACCACA]), M2 (a perfect match [GACCACA]), and M3 (putative Smad binding site [CAGACA]). Putative Runx2 and Smad binding sites are indicated by solid and dotted underlining, respectively, and mutated nucleotides are marked by asterisks. (B) EMSA was performed by using the TβRE probe and nuclear lysates obtained from C2C12 cells untreated or treated with TGF-β1 or BMP-2 for 24 h. The reactions were performed in the presence or absence of PEBP2β/Cbfβ protein. A 50-fold molar excess of unlabeled mutant oligonucleotide was incubated with the nuclear lysates as competitor DNA (Ct). The arrow and arrowhead indicate the TβRE binding complex and free probe, respectively. (C) EMSA was performed by using the same nuclear lysates and TβRE probes in the presence or absence of a polyclonal antibody which recognized all members of the Runx α subunit (anti-α; lane 2 and 5) or β subunit (anti-β; lane 3 and 6) or a monoclonal antibody which specifically recognized Runx2 (anti-Runx2; lanes 8, 10, and 12). The arrowheads and arrows indicate the positions of Runx2 and Runx-antibody complexes, respectively. (D) Time course induction of Runx2 was analyzed by EMSA using nuclear lysates prepared from cells treated with TGF-β1 for 0, 4, 12, 18, 24, 72, and 120 h. A nuclear extract prepared from cells cultured for 72 h in the absence of TGF-β1 was used as a control. EMSA was performed with the TβRE probe in the presence or absence of a 50-fold molar excess of unlabeled M3 as competitor. The arrow indicates the TβRE binding complex. Ct, competitor.

    Article Snippet: The mouse pluripotent mesenchymal precursor cell line C2C12 was purchased from the American Type Culture Collection C2C12 and MC3T3-E1 cells were maintained in Dulbecco modified Eagle medium (DMEM) containing 15% fetal bovine serum (FBS), and penicillin G (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere of 5% CO2 in air.

    Techniques: Mutagenesis, Binding Assay, Incubation, Cell Culture

    Induction of Runx2 mRNA by TGF-β1 and BMP2. (A) C2C12 cells were treated with TGF-β1 (5 ng/ml) or BMP-2 (300 ng/ml) for the indicated times, and total RNA was prepared. Northern blotting was performed using PEBP2αA ), which contains the common region of Runx2-αA1 and Runx2-til-1 , as a probe for Runx2 (Rx2-Ct). (B) Total RNAs were prepared from C2C12 cells treated with BMP-2 (300 ng/ml) for 6 h and analyzed by Northern blot hybridization using the Rx2-Ct probe. The same blot was stripped and rehybridized with Runx2-αA1 )- or Runx2-til-1 )-specific probes. A probe prepared from the GAPDH coding sequence was used as a loading control.

    Journal: Molecular and Cellular Biology

    Article Title: Runx2 Is a Common Target of Transforming Growth Factor ?1 and Bone Morphogenetic Protein 2, and Cooperation between Runx2 and Smad5 Induces Osteoblast-Specific Gene Expression in the Pluripotent Mesenchymal Precursor Cell Line C2C12

    doi:

    Figure Lengend Snippet: Induction of Runx2 mRNA by TGF-β1 and BMP2. (A) C2C12 cells were treated with TGF-β1 (5 ng/ml) or BMP-2 (300 ng/ml) for the indicated times, and total RNA was prepared. Northern blotting was performed using PEBP2αA ), which contains the common region of Runx2-αA1 and Runx2-til-1 , as a probe for Runx2 (Rx2-Ct). (B) Total RNAs were prepared from C2C12 cells treated with BMP-2 (300 ng/ml) for 6 h and analyzed by Northern blot hybridization using the Rx2-Ct probe. The same blot was stripped and rehybridized with Runx2-αA1 )- or Runx2-til-1 )-specific probes. A probe prepared from the GAPDH coding sequence was used as a loading control.

    Article Snippet: The mouse pluripotent mesenchymal precursor cell line C2C12 was purchased from the American Type Culture Collection C2C12 and MC3T3-E1 cells were maintained in Dulbecco modified Eagle medium (DMEM) containing 15% fetal bovine serum (FBS), and penicillin G (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere of 5% CO2 in air.

    Techniques: Northern Blot, Hybridization, Sequencing

    Binding of Runx2 to the Runx binding site is essential for the TGF-β1 responsiveness of the TβRE. (A) Diagrams of luciferase reporter constructs. Two reporters for the TβRE of the Ig Cα promoter were constructed using the pGL3-promoter plasmid (Promega) containing the simian virus 40 promoter (SV40 Pr) as a backbone, i.e., one with the wild-type (WT) TβRE (pGL3-TβRE) DNA and the other with the TβRE mutated at the Runx binding site (pGL3-M2). Two copies of the element were inserted for each plasmid construct. (B) pGL3, pGL3-M2, and pGL3-TβRE reporters were transfected into C2C12 cells with the Runx2 expression plasmid (Runx2) or the constitutively active TGF-β receptor I expression plasmid (TβR-I) or with both. Cells were harvested 48 h after transfection, and luciferase activities were assayed. (C and D) The same reporters were transfected into MC3T3-E1 and H1-127-30 cells with or without the Runx2 expression plasmid (Runx2) and cultured in the presence or absence of TGF-β1 for 24 h. Luciferase activities were measured, and relative activities are shown.

    Journal: Molecular and Cellular Biology

    Article Title: Runx2 Is a Common Target of Transforming Growth Factor ?1 and Bone Morphogenetic Protein 2, and Cooperation between Runx2 and Smad5 Induces Osteoblast-Specific Gene Expression in the Pluripotent Mesenchymal Precursor Cell Line C2C12

    doi:

    Figure Lengend Snippet: Binding of Runx2 to the Runx binding site is essential for the TGF-β1 responsiveness of the TβRE. (A) Diagrams of luciferase reporter constructs. Two reporters for the TβRE of the Ig Cα promoter were constructed using the pGL3-promoter plasmid (Promega) containing the simian virus 40 promoter (SV40 Pr) as a backbone, i.e., one with the wild-type (WT) TβRE (pGL3-TβRE) DNA and the other with the TβRE mutated at the Runx binding site (pGL3-M2). Two copies of the element were inserted for each plasmid construct. (B) pGL3, pGL3-M2, and pGL3-TβRE reporters were transfected into C2C12 cells with the Runx2 expression plasmid (Runx2) or the constitutively active TGF-β receptor I expression plasmid (TβR-I) or with both. Cells were harvested 48 h after transfection, and luciferase activities were assayed. (C and D) The same reporters were transfected into MC3T3-E1 and H1-127-30 cells with or without the Runx2 expression plasmid (Runx2) and cultured in the presence or absence of TGF-β1 for 24 h. Luciferase activities were measured, and relative activities are shown.

    Article Snippet: The mouse pluripotent mesenchymal precursor cell line C2C12 was purchased from the American Type Culture Collection C2C12 and MC3T3-E1 cells were maintained in Dulbecco modified Eagle medium (DMEM) containing 15% fetal bovine serum (FBS), and penicillin G (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere of 5% CO2 in air.

    Techniques: Binding Assay, Luciferase, Construct, Plasmid Preparation, Transfection, Expressing, Cell Culture

    Morphological changes of C2C12 cells stably expressing Runx2. (A) Western blotting showing overexpression of Runx2 in C2C12-Rx2 cells. Cytoplasmic (C) and nuclear (N) protein extracts were prepared from C2C12 and C2C12-Rx2 cells, and Runx2 protein was detected by Western blotting. An arrow indicates Runx2 protein. (B) C2C12 cells containing the empty vector were cultured for 10 days in differentiation medium (5% FBS in DMEM) in the presence or absence of TGF-β1 (5 ng/ml) or BMP-2 (300 ng/ml). C2C12-Rx2 cells were cultured under the same conditions in the absence of TGF-β1 and BMP-2. After incubation, morphological changes were compared.

    Journal: Molecular and Cellular Biology

    Article Title: Runx2 Is a Common Target of Transforming Growth Factor ?1 and Bone Morphogenetic Protein 2, and Cooperation between Runx2 and Smad5 Induces Osteoblast-Specific Gene Expression in the Pluripotent Mesenchymal Precursor Cell Line C2C12

    doi:

    Figure Lengend Snippet: Morphological changes of C2C12 cells stably expressing Runx2. (A) Western blotting showing overexpression of Runx2 in C2C12-Rx2 cells. Cytoplasmic (C) and nuclear (N) protein extracts were prepared from C2C12 and C2C12-Rx2 cells, and Runx2 protein was detected by Western blotting. An arrow indicates Runx2 protein. (B) C2C12 cells containing the empty vector were cultured for 10 days in differentiation medium (5% FBS in DMEM) in the presence or absence of TGF-β1 (5 ng/ml) or BMP-2 (300 ng/ml). C2C12-Rx2 cells were cultured under the same conditions in the absence of TGF-β1 and BMP-2. After incubation, morphological changes were compared.

    Article Snippet: The mouse pluripotent mesenchymal precursor cell line C2C12 was purchased from the American Type Culture Collection C2C12 and MC3T3-E1 cells were maintained in Dulbecco modified Eagle medium (DMEM) containing 15% fetal bovine serum (FBS), and penicillin G (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere of 5% CO2 in air.

    Techniques: Stable Transfection, Expressing, Western Blot, Over Expression, Plasmid Preparation, Cell Culture, Incubation

    Pattern of gene expression following BMP-2 and TGF-β1 treatment of C2C12 and C2C12-Rx2 cells. Control C2C12 (lane 1 to 3) and C2C12-Rx2 (lane 4 to 6) cells were treated with TGF-β1 (5 ng/ml) or BMP-2 (300 ng/ml) for 3 days. Total RNA was extracted and analyzed by Northern blotting with probes homologous to osteocalcin (OC), collagen type I (Col-I), fibronectin (FN), or MyoD.

    Journal: Molecular and Cellular Biology

    Article Title: Runx2 Is a Common Target of Transforming Growth Factor ?1 and Bone Morphogenetic Protein 2, and Cooperation between Runx2 and Smad5 Induces Osteoblast-Specific Gene Expression in the Pluripotent Mesenchymal Precursor Cell Line C2C12

    doi:

    Figure Lengend Snippet: Pattern of gene expression following BMP-2 and TGF-β1 treatment of C2C12 and C2C12-Rx2 cells. Control C2C12 (lane 1 to 3) and C2C12-Rx2 (lane 4 to 6) cells were treated with TGF-β1 (5 ng/ml) or BMP-2 (300 ng/ml) for 3 days. Total RNA was extracted and analyzed by Northern blotting with probes homologous to osteocalcin (OC), collagen type I (Col-I), fibronectin (FN), or MyoD.

    Article Snippet: The mouse pluripotent mesenchymal precursor cell line C2C12 was purchased from the American Type Culture Collection C2C12 and MC3T3-E1 cells were maintained in Dulbecco modified Eagle medium (DMEM) containing 15% fetal bovine serum (FBS), and penicillin G (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere of 5% CO2 in air.

    Techniques: Expressing, Northern Blot

    Tanshinol regulates expression of KLF15 gene under condition of Dex involving glucocorticoid receptor. C2C12 cells and MC3T3-E1 cells were treated with Dex and/or RU486 (RU, a direct target of glucocorticoid receptor) in the presence or absence of Tan for 12 h; mRNA expression of KLF15 gene was measured by qRT-PCR. Values are means ± SD of at least three independent experiments. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Tanshinol Rescues the Impaired Bone Formation Elicited by Glucocorticoid Involved in KLF15 Pathway

    doi: 10.1155/2016/1092746

    Figure Lengend Snippet: Tanshinol regulates expression of KLF15 gene under condition of Dex involving glucocorticoid receptor. C2C12 cells and MC3T3-E1 cells were treated with Dex and/or RU486 (RU, a direct target of glucocorticoid receptor) in the presence or absence of Tan for 12 h; mRNA expression of KLF15 gene was measured by qRT-PCR. Values are means ± SD of at least three independent experiments. ∗ P

    Article Snippet: Cell Culture and Osteoblastic Differentiation Assay The pluripotent mesenchymal precursor C2C12 cells and preosteoblastic MC3T3-E1 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques: Expressing, Quantitative RT-PCR

    Tanshinol attenuates downregulation of canonical Wnt signaling elicited by Dex associated with regulation KLF15. ((a) and (b)) C2C12 cells or MC3T3-E1 cells were cotransfected with the Tcf4-luc or FoxO3a-luc reporter plasmid in combination with KLF15 siRNA or the scrambled sequence. ((c) and (d)) C2C12 cells or MC3T3-E1 cells were infected with the FoxO3a-luc or Tcf4-luc reporter plasmid in combination with recombinant adenovirus Ad-KLF15 or mock (noninfection). Luciferase activity assays were explored using the Dual-Luciferase Reporter Assay System as described under Section 2.8 in Materials and Methods. The data represent mean ± SD of luciferase relative luminescence units (RLU) normalized to corresponding renilla luciferase activity (triplicates). (e) Tcf4 (a requisite mediator for downstream effector Tcf of canonical Wnt pathway contributing to bone formation) in MC3T3-E1 cells exposed to Ad-KLF15 and mock was detected by Western blot assay. Representative figure was shown on the left panel, and quantification is shown on the right panel. Bars indicate mean ± SD of triplicate determinations. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Tanshinol Rescues the Impaired Bone Formation Elicited by Glucocorticoid Involved in KLF15 Pathway

    doi: 10.1155/2016/1092746

    Figure Lengend Snippet: Tanshinol attenuates downregulation of canonical Wnt signaling elicited by Dex associated with regulation KLF15. ((a) and (b)) C2C12 cells or MC3T3-E1 cells were cotransfected with the Tcf4-luc or FoxO3a-luc reporter plasmid in combination with KLF15 siRNA or the scrambled sequence. ((c) and (d)) C2C12 cells or MC3T3-E1 cells were infected with the FoxO3a-luc or Tcf4-luc reporter plasmid in combination with recombinant adenovirus Ad-KLF15 or mock (noninfection). Luciferase activity assays were explored using the Dual-Luciferase Reporter Assay System as described under Section 2.8 in Materials and Methods. The data represent mean ± SD of luciferase relative luminescence units (RLU) normalized to corresponding renilla luciferase activity (triplicates). (e) Tcf4 (a requisite mediator for downstream effector Tcf of canonical Wnt pathway contributing to bone formation) in MC3T3-E1 cells exposed to Ad-KLF15 and mock was detected by Western blot assay. Representative figure was shown on the left panel, and quantification is shown on the right panel. Bars indicate mean ± SD of triplicate determinations. ∗ P

    Article Snippet: Cell Culture and Osteoblastic Differentiation Assay The pluripotent mesenchymal precursor C2C12 cells and preosteoblastic MC3T3-E1 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques: Plasmid Preparation, Sequencing, Infection, Recombinant, Luciferase, Activity Assay, Reporter Assay, Western Blot

    Tanshinol counteracts inhibition of osteoblastic differentiation and bone formation elicited by Dex in connection with downregulation of KLF15. C2C12 cells and MC3T3-E1 cells were transfected with KLF15 siRNA for 18 h, followed by DMEM medium supplemented with Dex in the presence or absence of tanshinol for 7 days. (a) Capacity of osteoblastic differentiation in C2C12 cells was determined by using ALP staining. (b) Capacity of osteoblastic differentiation in MC3T3-E1 cells. Original magnification (×100) in representative microscopic images. (c) Effects of knockdown of KLF15 on activity of bone formation. MC3T3-E1 cells were treated with KLF15 siRNA for 18 h, followed by Dex treatment with or without tanshinol for 21 days. Mineralization activity with the indicated treatments was stained using Alizarin Red S at day 21. Original magnification (×100) in representative microscopic images (upper panel). Quantitative determination was carried out by CPC solution (pH 7.0) (lower panel). Vehicle: vehicle control (Veh). Values are means ± SD of at least three independent experiments. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Tanshinol Rescues the Impaired Bone Formation Elicited by Glucocorticoid Involved in KLF15 Pathway

    doi: 10.1155/2016/1092746

    Figure Lengend Snippet: Tanshinol counteracts inhibition of osteoblastic differentiation and bone formation elicited by Dex in connection with downregulation of KLF15. C2C12 cells and MC3T3-E1 cells were transfected with KLF15 siRNA for 18 h, followed by DMEM medium supplemented with Dex in the presence or absence of tanshinol for 7 days. (a) Capacity of osteoblastic differentiation in C2C12 cells was determined by using ALP staining. (b) Capacity of osteoblastic differentiation in MC3T3-E1 cells. Original magnification (×100) in representative microscopic images. (c) Effects of knockdown of KLF15 on activity of bone formation. MC3T3-E1 cells were treated with KLF15 siRNA for 18 h, followed by Dex treatment with or without tanshinol for 21 days. Mineralization activity with the indicated treatments was stained using Alizarin Red S at day 21. Original magnification (×100) in representative microscopic images (upper panel). Quantitative determination was carried out by CPC solution (pH 7.0) (lower panel). Vehicle: vehicle control (Veh). Values are means ± SD of at least three independent experiments. ∗ P

    Article Snippet: Cell Culture and Osteoblastic Differentiation Assay The pluripotent mesenchymal precursor C2C12 cells and preosteoblastic MC3T3-E1 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques: Inhibition, Transfection, ALP Assay, Staining, Activity Assay