pluripotent mesenchymal precursor c2c12 cells  (ATCC)


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    C2C12
    Description:
    Applications transfection host Cell Type myoblast Host Mus musculus mouse
    Catalog Number:
    CRL-1772
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    Applications:
    transfection host
    Cell Type:
    myoblast
    Host:
    Mus musculus, mouse
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    ATCC pluripotent mesenchymal precursor c2c12 cells
    Tanshinol regulates expression of KLF15 gene under condition of Dex involving glucocorticoid receptor. <t>C2C12</t> 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
    Applications transfection host Cell Type myoblast Host Mus musculus mouse
    https://www.bioz.com/result/pluripotent mesenchymal precursor c2c12 cells/product/ATCC
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pluripotent mesenchymal precursor c2c12 cells - by Bioz Stars, 2021-06
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    Images

    1) Product Images from "Tanshinol Rescues the Impaired Bone Formation Elicited by Glucocorticoid Involved in KLF15 Pathway"

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

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2016/1092746

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

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

    2) Product Images from "Tanshinol Attenuates the Deleterious Effects of Oxidative Stress on Osteoblastic Differentiation via Wnt/FoxO3a Signaling"

    Article Title: Tanshinol Attenuates the Deleterious Effects of Oxidative Stress on Osteoblastic Differentiation via Wnt/FoxO3a Signaling

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2013/351895

    Tanshinol diminishes the cell cycle arrest and apoptosis under oxidative stress. C2C12 cells were pretreated with the indicated concentrations of Tanshinol or Resveratrol for 1 h, followed by the treatment of vehicle control or H 2 O 2 for 24 h. The following measurements were carried out. (a) Analysis of cell cycle distribution was explored using flow cytometry; (b) the activation of caspase 3 was determined by ELISA kit; (c) observations of morphological alterations of apoptosis cells were addressed by Hoechst33258 staining (left panel) and counted by Image J software (right panel); (d) the ultrastructural differences between apoptosis cells and normal cells were examined using TEM. Note: (1) Con (vehicle control); (2) H (H 2 O 2 ); (3) H + T (H 2 O 2 + Tanshinol); (4) H + R (H 2 O 2 + Resveratrol). Error bars indicate mean ± SEM of at least three independent experiments. * P
    Figure Legend Snippet: Tanshinol diminishes the cell cycle arrest and apoptosis under oxidative stress. C2C12 cells were pretreated with the indicated concentrations of Tanshinol or Resveratrol for 1 h, followed by the treatment of vehicle control or H 2 O 2 for 24 h. The following measurements were carried out. (a) Analysis of cell cycle distribution was explored using flow cytometry; (b) the activation of caspase 3 was determined by ELISA kit; (c) observations of morphological alterations of apoptosis cells were addressed by Hoechst33258 staining (left panel) and counted by Image J software (right panel); (d) the ultrastructural differences between apoptosis cells and normal cells were examined using TEM. Note: (1) Con (vehicle control); (2) H (H 2 O 2 ); (3) H + T (H 2 O 2 + Tanshinol); (4) H + R (H 2 O 2 + Resveratrol). Error bars indicate mean ± SEM of at least three independent experiments. * P

    Techniques Used: Flow Cytometry, Cytometry, Activation Assay, Enzyme-linked Immunosorbent Assay, Staining, Software, Transmission Electron Microscopy

    Tanshinol rescues oxidative stress-elicited inhibition of Wnt/ β -catenin signaling. (a) C2C12 cells were treated as described in Figure 4 , and protein levels of β -catenin were detected by Western Blot. Representative immunoblots were shown in upper panel. Quantitative results of relative band intensities of protein are showed in lower panel. (b) C2C12 cells were transfected with the Tcf-luc reporter plasmid or negative control. Cells transfected were treated with or without Tanshinol in the presence or absence of Wnt3a for 1 h, followed by vehicle control, H 2 O 2 for 24 h. Luciferase activity assays were explored as described under “ Section 2 ”. The data represent mean ± SEM of luciferase relative luminescence units (RLU) normalized to corresponding Renilla luciferase activity (triplicates). (c) C2C12 cells were treated as described in Figure 4 , and the expression levels of Axin2 , ALP , and OPG mRNA were quantified by qRT-PCR and normalized to GADPH mRNA. Note: (1) Con (vehicle control); (2) H (H 2 O 2 ); (3) T (Tanshinol); (4) H + T (H 2 O 2 + Tanshinol); (5) H + R (H 2 O 2 + Resveratrol). Error bars indicate mean ± SEM of at least three independent experiments. * P
    Figure Legend Snippet: Tanshinol rescues oxidative stress-elicited inhibition of Wnt/ β -catenin signaling. (a) C2C12 cells were treated as described in Figure 4 , and protein levels of β -catenin were detected by Western Blot. Representative immunoblots were shown in upper panel. Quantitative results of relative band intensities of protein are showed in lower panel. (b) C2C12 cells were transfected with the Tcf-luc reporter plasmid or negative control. Cells transfected were treated with or without Tanshinol in the presence or absence of Wnt3a for 1 h, followed by vehicle control, H 2 O 2 for 24 h. Luciferase activity assays were explored as described under “ Section 2 ”. The data represent mean ± SEM of luciferase relative luminescence units (RLU) normalized to corresponding Renilla luciferase activity (triplicates). (c) C2C12 cells were treated as described in Figure 4 , and the expression levels of Axin2 , ALP , and OPG mRNA were quantified by qRT-PCR and normalized to GADPH mRNA. Note: (1) Con (vehicle control); (2) H (H 2 O 2 ); (3) T (Tanshinol); (4) H + T (H 2 O 2 + Tanshinol); (5) H + R (H 2 O 2 + Resveratrol). Error bars indicate mean ± SEM of at least three independent experiments. * P

    Techniques Used: Inhibition, Western Blot, Transfection, Plasmid Preparation, Negative Control, Luciferase, Activity Assay, Expressing, ALP Assay, Quantitative RT-PCR

    Protective effects of Tanshinol against H 2 O 2 -induced cell death and ROS generation. (a) Cell viability was measured by MTT assay in C2C12 cells exposed to H 2 O 2 at the indicated concentration for 24 h. (b) C2C12 cells were pretreated in the presence or absence of Tanshinol (1 μ M) for 1 h before the addition of H 2 O 2 (200 μ M) and were subsequently cultured for 0 h, 12 h, 24 h, 36 h, and 48 h, respectively. (c) The chemical structure of Tanshinol consists of polyphenolic hydroxyl groups. (d) C2C12 cells were pretreated as described in (b), followed by H 2 O 2 (200 μ M) for 24 h, and the oxidative status of C2C12 cells was quantified by DCHF-DA to monitor the emitted fluorescence intensity resulting from intracellular oxidation using flow cytometry. Note: (1) Con (vehicle control); (2) H (H 2 O 2 ); (3) H + T (H 2 O 2 + Tanshinol); (4) H + R (H 2 O 2 + Resveratrol). Data are given as mean ± SEM of at least three independent experiments. * P
    Figure Legend Snippet: Protective effects of Tanshinol against H 2 O 2 -induced cell death and ROS generation. (a) Cell viability was measured by MTT assay in C2C12 cells exposed to H 2 O 2 at the indicated concentration for 24 h. (b) C2C12 cells were pretreated in the presence or absence of Tanshinol (1 μ M) for 1 h before the addition of H 2 O 2 (200 μ M) and were subsequently cultured for 0 h, 12 h, 24 h, 36 h, and 48 h, respectively. (c) The chemical structure of Tanshinol consists of polyphenolic hydroxyl groups. (d) C2C12 cells were pretreated as described in (b), followed by H 2 O 2 (200 μ M) for 24 h, and the oxidative status of C2C12 cells was quantified by DCHF-DA to monitor the emitted fluorescence intensity resulting from intracellular oxidation using flow cytometry. Note: (1) Con (vehicle control); (2) H (H 2 O 2 ); (3) H + T (H 2 O 2 + Tanshinol); (4) H + R (H 2 O 2 + Resveratrol). Data are given as mean ± SEM of at least three independent experiments. * P

    Techniques Used: MTT Assay, Concentration Assay, Cell Culture, Fluorescence, Flow Cytometry, Cytometry

    Effects of Tanshinol on Tcf- and FoxO3a-mediated transcription activity in C2C12 cells and MC3T3-E1 cells treated with FoxO3a siRNA, or with overexpression of β -Catenin or Tcf4 under oxidative stress. ((a), (c), and (e)) C2C12 cells or MC3T3-E1 cells were cotransfected with the FoxO3a-luc reporter plasmid in combination with FoxO3a siRNA, or pcDNA3- β -catenin, or pcDNA3-Tcf4, or corresponding control using Attractene transfection reagent. Other procedures were carried out as described in Figure 5(b) . ((b), (d), and (f)) Cells were cotransfected with Tcf-luc reporter plasmid and other treatment described in (a), (c), and (e). Others procedure were made as described in Figure 5(b) . Bars indicate mean ± SME of triplicate determinations. * P
    Figure Legend Snippet: Effects of Tanshinol on Tcf- and FoxO3a-mediated transcription activity in C2C12 cells and MC3T3-E1 cells treated with FoxO3a siRNA, or with overexpression of β -Catenin or Tcf4 under oxidative stress. ((a), (c), and (e)) C2C12 cells or MC3T3-E1 cells were cotransfected with the FoxO3a-luc reporter plasmid in combination with FoxO3a siRNA, or pcDNA3- β -catenin, or pcDNA3-Tcf4, or corresponding control using Attractene transfection reagent. Other procedures were carried out as described in Figure 5(b) . ((b), (d), and (f)) Cells were cotransfected with Tcf-luc reporter plasmid and other treatment described in (a), (c), and (e). Others procedure were made as described in Figure 5(b) . Bars indicate mean ± SME of triplicate determinations. * P

    Techniques Used: Activity Assay, Over Expression, Plasmid Preparation, Transfection

    Effects of Tanshinol on targets genes of Wnt/Tcf and FoxO3a in C2C12 cells and MC3T3-E1 cells treated with FoxO3a siRNA, or with Tcf4 overexpression under oxidative stress. ((a), (c), (e), and (g)) C2C12 cells or MC3T3-E1 cells were transfected transiently with either the FoxO3a siRNA or the scrambled RNA control in the presence or absence of vehicle, H 2 O 2 , or Tashinol for 24 hours. The relative mRNA expression levels of Gadd45a , CAT , Axin2 , and ALP genes in control cells (Scrambled) and in the FoxO3a-knockdown (FoxO3a siRNA) cells were assessed by quantitative RT-PCR assay. ((b), (d), (f), and (h)) C2C12 cells or MC3T3-E1 cells were transfected transiently with either the overexpression plasmid of Tcf4 or the empty vector control. The following procedure was executed as described in (a), (c), (e), and (g). mRNA values were normalized to GAPDH mRNA. Bars represent mean ± SME of three independent experiments. * P
    Figure Legend Snippet: Effects of Tanshinol on targets genes of Wnt/Tcf and FoxO3a in C2C12 cells and MC3T3-E1 cells treated with FoxO3a siRNA, or with Tcf4 overexpression under oxidative stress. ((a), (c), (e), and (g)) C2C12 cells or MC3T3-E1 cells were transfected transiently with either the FoxO3a siRNA or the scrambled RNA control in the presence or absence of vehicle, H 2 O 2 , or Tashinol for 24 hours. The relative mRNA expression levels of Gadd45a , CAT , Axin2 , and ALP genes in control cells (Scrambled) and in the FoxO3a-knockdown (FoxO3a siRNA) cells were assessed by quantitative RT-PCR assay. ((b), (d), (f), and (h)) C2C12 cells or MC3T3-E1 cells were transfected transiently with either the overexpression plasmid of Tcf4 or the empty vector control. The following procedure was executed as described in (a), (c), (e), and (g). mRNA values were normalized to GAPDH mRNA. Bars represent mean ± SME of three independent experiments. * P

    Techniques Used: Over Expression, Transfection, Expressing, ALP Assay, Quantitative RT-PCR, Plasmid Preparation

    Tanshinol counteracts inhibitory effect of oxidative stress on osteoblastic differentiation. (a) C2C12 cells were treated with increasing concentrations of BMP-2 for 24 h to determine the optimal concentration using qRT-PCR. Cells were cotreated with BMP-2 (100 ng/mL) and Tanshinol (1 μ M) or Resveratrol (1 μ M) in the presence or absence of H 2 O 2 (200 μ M), and determinations were made as follows. (b) Cells were stained using ALP staining Kit, and (c) the percentage of positively stained cell populations was counted by Image J software to analyse the extent of osteoblastic differentiation at day 3; (d) ALP activity in the cell lysates and (e) osteocalcin secretion in the supernatant of media were measured by ALP Assay Kit and ELISA Kit at day 3, respectively; (f) mineralization activity with the indicated treatments was stained using alizarin red S at day 10. Note: (1) Con (vehicle control); (2) B (BMP-2); (3) B + T (BMP-2 + Tanshinol); (4) B + H (BMP-2 + H 2 O 2 ); (5) B + H + T (BMP-2 + H 2 O 2 + Tanshinol); (6) B + H + R (BMP-2 + H 2 O 2 + Resveratrol). Data shown are the mean ± SEM of at least three independent experiments. * P
    Figure Legend Snippet: Tanshinol counteracts inhibitory effect of oxidative stress on osteoblastic differentiation. (a) C2C12 cells were treated with increasing concentrations of BMP-2 for 24 h to determine the optimal concentration using qRT-PCR. Cells were cotreated with BMP-2 (100 ng/mL) and Tanshinol (1 μ M) or Resveratrol (1 μ M) in the presence or absence of H 2 O 2 (200 μ M), and determinations were made as follows. (b) Cells were stained using ALP staining Kit, and (c) the percentage of positively stained cell populations was counted by Image J software to analyse the extent of osteoblastic differentiation at day 3; (d) ALP activity in the cell lysates and (e) osteocalcin secretion in the supernatant of media were measured by ALP Assay Kit and ELISA Kit at day 3, respectively; (f) mineralization activity with the indicated treatments was stained using alizarin red S at day 10. Note: (1) Con (vehicle control); (2) B (BMP-2); (3) B + T (BMP-2 + Tanshinol); (4) B + H (BMP-2 + H 2 O 2 ); (5) B + H + T (BMP-2 + H 2 O 2 + Tanshinol); (6) B + H + R (BMP-2 + H 2 O 2 + Resveratrol). Data shown are the mean ± SEM of at least three independent experiments. * P

    Techniques Used: Concentration Assay, Quantitative RT-PCR, Staining, ALP Assay, Software, Activity Assay, Enzyme-linked Immunosorbent Assay

    Effects of Tanshinol on cell viability of C2C12 cells and MC3T3-E1 cells. (a) Cell viability was measured by MTT assay in pluripotent mesenchymal precursor C2C12 cells and (b) preosteoblastic MC3T3-E1 cells treated with or without the indicated concentrations of Tanshinol for 24 h. Data are given as mean ± SEM of at least three independent experiments. * P
    Figure Legend Snippet: Effects of Tanshinol on cell viability of C2C12 cells and MC3T3-E1 cells. (a) Cell viability was measured by MTT assay in pluripotent mesenchymal precursor C2C12 cells and (b) preosteoblastic MC3T3-E1 cells treated with or without the indicated concentrations of Tanshinol for 24 h. Data are given as mean ± SEM of at least three independent experiments. * P

    Techniques Used: MTT Assay

    Tanshinol attenuates the activation of FoxO3a in response to oxidative stress. (a) C2C12 cells were treated as described in Figure 4 , and protein expressions from whole cell lysates and nuclear fractions were immunoblotted for FoxO3a, β -actin (whole lysates marker), and Histone H3 (nuclear marker). Representative immunoblots were shown in upper panel. Quantitative analysis of relative band intensities of protein was showed in lower panel. (b and c) Cells were transfected with the FoxO3a-luc reporter plasmid, and the treatment of Wnt3a was replaced with LY294002 (50 μ M), LiCl (10 mM), or Dkk1 (500 ng/mL), and other procedures of experiments were addressed as described in Figure 5(b) . (d) C2C12 cells were treated as described in Figure 4 , and the expression levels of Gadd45 and CAT mRNA were quantified by qRT-PCR and normalized to GADPH mRNA. Note: (1) Con (vehicle control); (2) H (H 2 O 2 ); (3) T (Tanshinol); (4) H + T (H 2 O 2 + Tanshinol); (5) H + R (H 2 O 2 + Resveratrol). Error bars indicate mean ± SEM of at least three independent experiments. * P
    Figure Legend Snippet: Tanshinol attenuates the activation of FoxO3a in response to oxidative stress. (a) C2C12 cells were treated as described in Figure 4 , and protein expressions from whole cell lysates and nuclear fractions were immunoblotted for FoxO3a, β -actin (whole lysates marker), and Histone H3 (nuclear marker). Representative immunoblots were shown in upper panel. Quantitative analysis of relative band intensities of protein was showed in lower panel. (b and c) Cells were transfected with the FoxO3a-luc reporter plasmid, and the treatment of Wnt3a was replaced with LY294002 (50 μ M), LiCl (10 mM), or Dkk1 (500 ng/mL), and other procedures of experiments were addressed as described in Figure 5(b) . (d) C2C12 cells were treated as described in Figure 4 , and the expression levels of Gadd45 and CAT mRNA were quantified by qRT-PCR and normalized to GADPH mRNA. Note: (1) Con (vehicle control); (2) H (H 2 O 2 ); (3) T (Tanshinol); (4) H + T (H 2 O 2 + Tanshinol); (5) H + R (H 2 O 2 + Resveratrol). Error bars indicate mean ± SEM of at least three independent experiments. * P

    Techniques Used: Activation Assay, Marker, Western Blot, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR

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    ATCC pluripotent mesenchymal precursor c2c12 cells
    Tanshinol regulates expression of KLF15 gene under condition of Dex involving glucocorticoid receptor. <t>C2C12</t> 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
    Pluripotent Mesenchymal Precursor C2c12 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    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 diminishes the cell cycle arrest and apoptosis under oxidative stress. C2C12 cells were pretreated with the indicated concentrations of Tanshinol or Resveratrol for 1 h, followed by the treatment of vehicle control or H 2 O 2 for 24 h. The following measurements were carried out. (a) Analysis of cell cycle distribution was explored using flow cytometry; (b) the activation of caspase 3 was determined by ELISA kit; (c) observations of morphological alterations of apoptosis cells were addressed by Hoechst33258 staining (left panel) and counted by Image J software (right panel); (d) the ultrastructural differences between apoptosis cells and normal cells were examined using TEM. Note: (1) Con (vehicle control); (2) H (H 2 O 2 ); (3) H + T (H 2 O 2 + Tanshinol); (4) H + R (H 2 O 2 + Resveratrol). Error bars indicate mean ± SEM of at least three independent experiments. * P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Tanshinol Attenuates the Deleterious Effects of Oxidative Stress on Osteoblastic Differentiation via Wnt/FoxO3a Signaling

    doi: 10.1155/2013/351895

    Figure Lengend Snippet: Tanshinol diminishes the cell cycle arrest and apoptosis under oxidative stress. C2C12 cells were pretreated with the indicated concentrations of Tanshinol or Resveratrol for 1 h, followed by the treatment of vehicle control or H 2 O 2 for 24 h. The following measurements were carried out. (a) Analysis of cell cycle distribution was explored using flow cytometry; (b) the activation of caspase 3 was determined by ELISA kit; (c) observations of morphological alterations of apoptosis cells were addressed by Hoechst33258 staining (left panel) and counted by Image J software (right panel); (d) the ultrastructural differences between apoptosis cells and normal cells were examined using TEM. Note: (1) Con (vehicle control); (2) H (H 2 O 2 ); (3) H + T (H 2 O 2 + Tanshinol); (4) H + R (H 2 O 2 + Resveratrol). Error bars indicate mean ± SEM of at least three independent experiments. * P

    Article Snippet: Cell Culture, Transfection, and Luciferase Activity The pluripotent mesenchymal precursor C2C12 cells and preosteoblastic MC3T3-E1 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) and α -modified Eagle's medium (α -DMEM), respectively.

    Techniques: Flow Cytometry, Cytometry, Activation Assay, Enzyme-linked Immunosorbent Assay, Staining, Software, Transmission Electron Microscopy

    Tanshinol rescues oxidative stress-elicited inhibition of Wnt/ β -catenin signaling. (a) C2C12 cells were treated as described in Figure 4 , and protein levels of β -catenin were detected by Western Blot. Representative immunoblots were shown in upper panel. Quantitative results of relative band intensities of protein are showed in lower panel. (b) C2C12 cells were transfected with the Tcf-luc reporter plasmid or negative control. Cells transfected were treated with or without Tanshinol in the presence or absence of Wnt3a for 1 h, followed by vehicle control, H 2 O 2 for 24 h. Luciferase activity assays were explored as described under “ Section 2 ”. The data represent mean ± SEM of luciferase relative luminescence units (RLU) normalized to corresponding Renilla luciferase activity (triplicates). (c) C2C12 cells were treated as described in Figure 4 , and the expression levels of Axin2 , ALP , and OPG mRNA were quantified by qRT-PCR and normalized to GADPH mRNA. Note: (1) Con (vehicle control); (2) H (H 2 O 2 ); (3) T (Tanshinol); (4) H + T (H 2 O 2 + Tanshinol); (5) H + R (H 2 O 2 + Resveratrol). Error bars indicate mean ± SEM of at least three independent experiments. * P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Tanshinol Attenuates the Deleterious Effects of Oxidative Stress on Osteoblastic Differentiation via Wnt/FoxO3a Signaling

    doi: 10.1155/2013/351895

    Figure Lengend Snippet: Tanshinol rescues oxidative stress-elicited inhibition of Wnt/ β -catenin signaling. (a) C2C12 cells were treated as described in Figure 4 , and protein levels of β -catenin were detected by Western Blot. Representative immunoblots were shown in upper panel. Quantitative results of relative band intensities of protein are showed in lower panel. (b) C2C12 cells were transfected with the Tcf-luc reporter plasmid or negative control. Cells transfected were treated with or without Tanshinol in the presence or absence of Wnt3a for 1 h, followed by vehicle control, H 2 O 2 for 24 h. Luciferase activity assays were explored as described under “ Section 2 ”. The data represent mean ± SEM of luciferase relative luminescence units (RLU) normalized to corresponding Renilla luciferase activity (triplicates). (c) C2C12 cells were treated as described in Figure 4 , and the expression levels of Axin2 , ALP , and OPG mRNA were quantified by qRT-PCR and normalized to GADPH mRNA. Note: (1) Con (vehicle control); (2) H (H 2 O 2 ); (3) T (Tanshinol); (4) H + T (H 2 O 2 + Tanshinol); (5) H + R (H 2 O 2 + Resveratrol). Error bars indicate mean ± SEM of at least three independent experiments. * P

    Article Snippet: Cell Culture, Transfection, and Luciferase Activity The pluripotent mesenchymal precursor C2C12 cells and preosteoblastic MC3T3-E1 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) and α -modified Eagle's medium (α -DMEM), respectively.

    Techniques: Inhibition, Western Blot, Transfection, Plasmid Preparation, Negative Control, Luciferase, Activity Assay, Expressing, ALP Assay, Quantitative RT-PCR

    Protective effects of Tanshinol against H 2 O 2 -induced cell death and ROS generation. (a) Cell viability was measured by MTT assay in C2C12 cells exposed to H 2 O 2 at the indicated concentration for 24 h. (b) C2C12 cells were pretreated in the presence or absence of Tanshinol (1 μ M) for 1 h before the addition of H 2 O 2 (200 μ M) and were subsequently cultured for 0 h, 12 h, 24 h, 36 h, and 48 h, respectively. (c) The chemical structure of Tanshinol consists of polyphenolic hydroxyl groups. (d) C2C12 cells were pretreated as described in (b), followed by H 2 O 2 (200 μ M) for 24 h, and the oxidative status of C2C12 cells was quantified by DCHF-DA to monitor the emitted fluorescence intensity resulting from intracellular oxidation using flow cytometry. Note: (1) Con (vehicle control); (2) H (H 2 O 2 ); (3) H + T (H 2 O 2 + Tanshinol); (4) H + R (H 2 O 2 + Resveratrol). Data are given as mean ± SEM of at least three independent experiments. * P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Tanshinol Attenuates the Deleterious Effects of Oxidative Stress on Osteoblastic Differentiation via Wnt/FoxO3a Signaling

    doi: 10.1155/2013/351895

    Figure Lengend Snippet: Protective effects of Tanshinol against H 2 O 2 -induced cell death and ROS generation. (a) Cell viability was measured by MTT assay in C2C12 cells exposed to H 2 O 2 at the indicated concentration for 24 h. (b) C2C12 cells were pretreated in the presence or absence of Tanshinol (1 μ M) for 1 h before the addition of H 2 O 2 (200 μ M) and were subsequently cultured for 0 h, 12 h, 24 h, 36 h, and 48 h, respectively. (c) The chemical structure of Tanshinol consists of polyphenolic hydroxyl groups. (d) C2C12 cells were pretreated as described in (b), followed by H 2 O 2 (200 μ M) for 24 h, and the oxidative status of C2C12 cells was quantified by DCHF-DA to monitor the emitted fluorescence intensity resulting from intracellular oxidation using flow cytometry. Note: (1) Con (vehicle control); (2) H (H 2 O 2 ); (3) H + T (H 2 O 2 + Tanshinol); (4) H + R (H 2 O 2 + Resveratrol). Data are given as mean ± SEM of at least three independent experiments. * P

    Article Snippet: Cell Culture, Transfection, and Luciferase Activity The pluripotent mesenchymal precursor C2C12 cells and preosteoblastic MC3T3-E1 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) and α -modified Eagle's medium (α -DMEM), respectively.

    Techniques: MTT Assay, Concentration Assay, Cell Culture, Fluorescence, Flow Cytometry, Cytometry

    Effects of Tanshinol on Tcf- and FoxO3a-mediated transcription activity in C2C12 cells and MC3T3-E1 cells treated with FoxO3a siRNA, or with overexpression of β -Catenin or Tcf4 under oxidative stress. ((a), (c), and (e)) C2C12 cells or MC3T3-E1 cells were cotransfected with the FoxO3a-luc reporter plasmid in combination with FoxO3a siRNA, or pcDNA3- β -catenin, or pcDNA3-Tcf4, or corresponding control using Attractene transfection reagent. Other procedures were carried out as described in Figure 5(b) . ((b), (d), and (f)) Cells were cotransfected with Tcf-luc reporter plasmid and other treatment described in (a), (c), and (e). Others procedure were made as described in Figure 5(b) . Bars indicate mean ± SME of triplicate determinations. * P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Tanshinol Attenuates the Deleterious Effects of Oxidative Stress on Osteoblastic Differentiation via Wnt/FoxO3a Signaling

    doi: 10.1155/2013/351895

    Figure Lengend Snippet: Effects of Tanshinol on Tcf- and FoxO3a-mediated transcription activity in C2C12 cells and MC3T3-E1 cells treated with FoxO3a siRNA, or with overexpression of β -Catenin or Tcf4 under oxidative stress. ((a), (c), and (e)) C2C12 cells or MC3T3-E1 cells were cotransfected with the FoxO3a-luc reporter plasmid in combination with FoxO3a siRNA, or pcDNA3- β -catenin, or pcDNA3-Tcf4, or corresponding control using Attractene transfection reagent. Other procedures were carried out as described in Figure 5(b) . ((b), (d), and (f)) Cells were cotransfected with Tcf-luc reporter plasmid and other treatment described in (a), (c), and (e). Others procedure were made as described in Figure 5(b) . Bars indicate mean ± SME of triplicate determinations. * P

    Article Snippet: Cell Culture, Transfection, and Luciferase Activity The pluripotent mesenchymal precursor C2C12 cells and preosteoblastic MC3T3-E1 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) and α -modified Eagle's medium (α -DMEM), respectively.

    Techniques: Activity Assay, Over Expression, Plasmid Preparation, Transfection

    Effects of Tanshinol on targets genes of Wnt/Tcf and FoxO3a in C2C12 cells and MC3T3-E1 cells treated with FoxO3a siRNA, or with Tcf4 overexpression under oxidative stress. ((a), (c), (e), and (g)) C2C12 cells or MC3T3-E1 cells were transfected transiently with either the FoxO3a siRNA or the scrambled RNA control in the presence or absence of vehicle, H 2 O 2 , or Tashinol for 24 hours. The relative mRNA expression levels of Gadd45a , CAT , Axin2 , and ALP genes in control cells (Scrambled) and in the FoxO3a-knockdown (FoxO3a siRNA) cells were assessed by quantitative RT-PCR assay. ((b), (d), (f), and (h)) C2C12 cells or MC3T3-E1 cells were transfected transiently with either the overexpression plasmid of Tcf4 or the empty vector control. The following procedure was executed as described in (a), (c), (e), and (g). mRNA values were normalized to GAPDH mRNA. Bars represent mean ± SME of three independent experiments. * P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Tanshinol Attenuates the Deleterious Effects of Oxidative Stress on Osteoblastic Differentiation via Wnt/FoxO3a Signaling

    doi: 10.1155/2013/351895

    Figure Lengend Snippet: Effects of Tanshinol on targets genes of Wnt/Tcf and FoxO3a in C2C12 cells and MC3T3-E1 cells treated with FoxO3a siRNA, or with Tcf4 overexpression under oxidative stress. ((a), (c), (e), and (g)) C2C12 cells or MC3T3-E1 cells were transfected transiently with either the FoxO3a siRNA or the scrambled RNA control in the presence or absence of vehicle, H 2 O 2 , or Tashinol for 24 hours. The relative mRNA expression levels of Gadd45a , CAT , Axin2 , and ALP genes in control cells (Scrambled) and in the FoxO3a-knockdown (FoxO3a siRNA) cells were assessed by quantitative RT-PCR assay. ((b), (d), (f), and (h)) C2C12 cells or MC3T3-E1 cells were transfected transiently with either the overexpression plasmid of Tcf4 or the empty vector control. The following procedure was executed as described in (a), (c), (e), and (g). mRNA values were normalized to GAPDH mRNA. Bars represent mean ± SME of three independent experiments. * P

    Article Snippet: Cell Culture, Transfection, and Luciferase Activity The pluripotent mesenchymal precursor C2C12 cells and preosteoblastic MC3T3-E1 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) and α -modified Eagle's medium (α -DMEM), respectively.

    Techniques: Over Expression, Transfection, Expressing, ALP Assay, Quantitative RT-PCR, Plasmid Preparation

    Tanshinol counteracts inhibitory effect of oxidative stress on osteoblastic differentiation. (a) C2C12 cells were treated with increasing concentrations of BMP-2 for 24 h to determine the optimal concentration using qRT-PCR. Cells were cotreated with BMP-2 (100 ng/mL) and Tanshinol (1 μ M) or Resveratrol (1 μ M) in the presence or absence of H 2 O 2 (200 μ M), and determinations were made as follows. (b) Cells were stained using ALP staining Kit, and (c) the percentage of positively stained cell populations was counted by Image J software to analyse the extent of osteoblastic differentiation at day 3; (d) ALP activity in the cell lysates and (e) osteocalcin secretion in the supernatant of media were measured by ALP Assay Kit and ELISA Kit at day 3, respectively; (f) mineralization activity with the indicated treatments was stained using alizarin red S at day 10. Note: (1) Con (vehicle control); (2) B (BMP-2); (3) B + T (BMP-2 + Tanshinol); (4) B + H (BMP-2 + H 2 O 2 ); (5) B + H + T (BMP-2 + H 2 O 2 + Tanshinol); (6) B + H + R (BMP-2 + H 2 O 2 + Resveratrol). Data shown are the mean ± SEM of at least three independent experiments. * P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Tanshinol Attenuates the Deleterious Effects of Oxidative Stress on Osteoblastic Differentiation via Wnt/FoxO3a Signaling

    doi: 10.1155/2013/351895

    Figure Lengend Snippet: Tanshinol counteracts inhibitory effect of oxidative stress on osteoblastic differentiation. (a) C2C12 cells were treated with increasing concentrations of BMP-2 for 24 h to determine the optimal concentration using qRT-PCR. Cells were cotreated with BMP-2 (100 ng/mL) and Tanshinol (1 μ M) or Resveratrol (1 μ M) in the presence or absence of H 2 O 2 (200 μ M), and determinations were made as follows. (b) Cells were stained using ALP staining Kit, and (c) the percentage of positively stained cell populations was counted by Image J software to analyse the extent of osteoblastic differentiation at day 3; (d) ALP activity in the cell lysates and (e) osteocalcin secretion in the supernatant of media were measured by ALP Assay Kit and ELISA Kit at day 3, respectively; (f) mineralization activity with the indicated treatments was stained using alizarin red S at day 10. Note: (1) Con (vehicle control); (2) B (BMP-2); (3) B + T (BMP-2 + Tanshinol); (4) B + H (BMP-2 + H 2 O 2 ); (5) B + H + T (BMP-2 + H 2 O 2 + Tanshinol); (6) B + H + R (BMP-2 + H 2 O 2 + Resveratrol). Data shown are the mean ± SEM of at least three independent experiments. * P

    Article Snippet: Cell Culture, Transfection, and Luciferase Activity The pluripotent mesenchymal precursor C2C12 cells and preosteoblastic MC3T3-E1 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) and α -modified Eagle's medium (α -DMEM), respectively.

    Techniques: Concentration Assay, Quantitative RT-PCR, Staining, ALP Assay, Software, Activity Assay, Enzyme-linked Immunosorbent Assay

    Effects of Tanshinol on cell viability of C2C12 cells and MC3T3-E1 cells. (a) Cell viability was measured by MTT assay in pluripotent mesenchymal precursor C2C12 cells and (b) preosteoblastic MC3T3-E1 cells treated with or without the indicated concentrations of Tanshinol for 24 h. Data are given as mean ± SEM of at least three independent experiments. * P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Tanshinol Attenuates the Deleterious Effects of Oxidative Stress on Osteoblastic Differentiation via Wnt/FoxO3a Signaling

    doi: 10.1155/2013/351895

    Figure Lengend Snippet: Effects of Tanshinol on cell viability of C2C12 cells and MC3T3-E1 cells. (a) Cell viability was measured by MTT assay in pluripotent mesenchymal precursor C2C12 cells and (b) preosteoblastic MC3T3-E1 cells treated with or without the indicated concentrations of Tanshinol for 24 h. Data are given as mean ± SEM of at least three independent experiments. * P

    Article Snippet: Cell Culture, Transfection, and Luciferase Activity The pluripotent mesenchymal precursor C2C12 cells and preosteoblastic MC3T3-E1 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) and α -modified Eagle's medium (α -DMEM), respectively.

    Techniques: MTT Assay

    Tanshinol attenuates the activation of FoxO3a in response to oxidative stress. (a) C2C12 cells were treated as described in Figure 4 , and protein expressions from whole cell lysates and nuclear fractions were immunoblotted for FoxO3a, β -actin (whole lysates marker), and Histone H3 (nuclear marker). Representative immunoblots were shown in upper panel. Quantitative analysis of relative band intensities of protein was showed in lower panel. (b and c) Cells were transfected with the FoxO3a-luc reporter plasmid, and the treatment of Wnt3a was replaced with LY294002 (50 μ M), LiCl (10 mM), or Dkk1 (500 ng/mL), and other procedures of experiments were addressed as described in Figure 5(b) . (d) C2C12 cells were treated as described in Figure 4 , and the expression levels of Gadd45 and CAT mRNA were quantified by qRT-PCR and normalized to GADPH mRNA. Note: (1) Con (vehicle control); (2) H (H 2 O 2 ); (3) T (Tanshinol); (4) H + T (H 2 O 2 + Tanshinol); (5) H + R (H 2 O 2 + Resveratrol). Error bars indicate mean ± SEM of at least three independent experiments. * P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Tanshinol Attenuates the Deleterious Effects of Oxidative Stress on Osteoblastic Differentiation via Wnt/FoxO3a Signaling

    doi: 10.1155/2013/351895

    Figure Lengend Snippet: Tanshinol attenuates the activation of FoxO3a in response to oxidative stress. (a) C2C12 cells were treated as described in Figure 4 , and protein expressions from whole cell lysates and nuclear fractions were immunoblotted for FoxO3a, β -actin (whole lysates marker), and Histone H3 (nuclear marker). Representative immunoblots were shown in upper panel. Quantitative analysis of relative band intensities of protein was showed in lower panel. (b and c) Cells were transfected with the FoxO3a-luc reporter plasmid, and the treatment of Wnt3a was replaced with LY294002 (50 μ M), LiCl (10 mM), or Dkk1 (500 ng/mL), and other procedures of experiments were addressed as described in Figure 5(b) . (d) C2C12 cells were treated as described in Figure 4 , and the expression levels of Gadd45 and CAT mRNA were quantified by qRT-PCR and normalized to GADPH mRNA. Note: (1) Con (vehicle control); (2) H (H 2 O 2 ); (3) T (Tanshinol); (4) H + T (H 2 O 2 + Tanshinol); (5) H + R (H 2 O 2 + Resveratrol). Error bars indicate mean ± SEM of at least three independent experiments. * P

    Article Snippet: Cell Culture, Transfection, and Luciferase Activity The pluripotent mesenchymal precursor C2C12 cells and preosteoblastic MC3T3-E1 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) and α -modified Eagle's medium (α -DMEM), respectively.

    Techniques: Activation Assay, Marker, Western Blot, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR

    Ectopic expression of DKK1 diminished Wnt3a induced OPG mRNA and protein in osteoblast cells. The expression of DKK family members in C2C12 (A) and human osteoblast cell lines (B) as determined by RT-PCR analysis are presented. Concentration of DKK1 protein

    Journal:

    Article Title: Myeloma-derived Dickkopf-1 disrupts Wnt-regulated osteoprotegerin and RANKL production by osteoblasts: a potential mechanism underlying osteolytic bone lesions in multiple myeloma

    doi: 10.1182/blood-2008-01-132134

    Figure Lengend Snippet: Ectopic expression of DKK1 diminished Wnt3a induced OPG mRNA and protein in osteoblast cells. The expression of DKK family members in C2C12 (A) and human osteoblast cell lines (B) as determined by RT-PCR analysis are presented. Concentration of DKK1 protein

    Article Snippet: Mouse pluripotent mesenchymal precursor cell line C2C12 that has the potential of differentiating into osteoblast in the presence of BMP-2 and human osteoblast cell line hFOB1.19 were purchased from ATCC (Manassas, VA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Concentration Assay

    DKK-1 inhibition of Wnt3a induced OPG mRNA and protein in osteoblast cells. C2C12 (A) and Saos-2 (B) cells were stimulated with or without Wnt3a for 8 hours after prior treatment with recombinant DKK-1 for 1 hour at indicated concentrations and then lysed.

    Journal:

    Article Title: Myeloma-derived Dickkopf-1 disrupts Wnt-regulated osteoprotegerin and RANKL production by osteoblasts: a potential mechanism underlying osteolytic bone lesions in multiple myeloma

    doi: 10.1182/blood-2008-01-132134

    Figure Lengend Snippet: DKK-1 inhibition of Wnt3a induced OPG mRNA and protein in osteoblast cells. C2C12 (A) and Saos-2 (B) cells were stimulated with or without Wnt3a for 8 hours after prior treatment with recombinant DKK-1 for 1 hour at indicated concentrations and then lysed.

    Article Snippet: Mouse pluripotent mesenchymal precursor cell line C2C12 that has the potential of differentiating into osteoblast in the presence of BMP-2 and human osteoblast cell line hFOB1.19 were purchased from ATCC (Manassas, VA).

    Techniques: Inhibition, Recombinant

    DKK1 and sera from MM patients inhibits Wnt3a-induced suppression of RANKL in osteoblast. C2C12 (A), Saos-2 (B), and MG63 (C) cells were treated with rWnt3a or Wnt3a-CM (as indicated) or Cont-CM for 48 hours after prior treatment with 100 ng/mL of DKK1

    Journal:

    Article Title: Myeloma-derived Dickkopf-1 disrupts Wnt-regulated osteoprotegerin and RANKL production by osteoblasts: a potential mechanism underlying osteolytic bone lesions in multiple myeloma

    doi: 10.1182/blood-2008-01-132134

    Figure Lengend Snippet: DKK1 and sera from MM patients inhibits Wnt3a-induced suppression of RANKL in osteoblast. C2C12 (A), Saos-2 (B), and MG63 (C) cells were treated with rWnt3a or Wnt3a-CM (as indicated) or Cont-CM for 48 hours after prior treatment with 100 ng/mL of DKK1

    Article Snippet: Mouse pluripotent mesenchymal precursor cell line C2C12 that has the potential of differentiating into osteoblast in the presence of BMP-2 and human osteoblast cell line hFOB1.19 were purchased from ATCC (Manassas, VA).

    Techniques:

    Knockdown DKK1 by shRNA restored Wnt3a-induced OPG in osteoblasts. C2C12 cells were transiently infected with supernatant containing control siRNA (shCont) or shRNA specific for DKK1 for indicated times. Total RNA was then isolated and subjected to RT-PCR

    Journal:

    Article Title: Myeloma-derived Dickkopf-1 disrupts Wnt-regulated osteoprotegerin and RANKL production by osteoblasts: a potential mechanism underlying osteolytic bone lesions in multiple myeloma

    doi: 10.1182/blood-2008-01-132134

    Figure Lengend Snippet: Knockdown DKK1 by shRNA restored Wnt3a-induced OPG in osteoblasts. C2C12 cells were transiently infected with supernatant containing control siRNA (shCont) or shRNA specific for DKK1 for indicated times. Total RNA was then isolated and subjected to RT-PCR

    Article Snippet: Mouse pluripotent mesenchymal precursor cell line C2C12 that has the potential of differentiating into osteoblast in the presence of BMP-2 and human osteoblast cell line hFOB1.19 were purchased from ATCC (Manassas, VA).

    Techniques: shRNA, Infection, Isolation, Reverse Transcription Polymerase Chain Reaction

    Neutralization of DKK1 rescued OPG expression in osteoblasts grown in the presence of MM sera or primary MM cells. C2C12 cells were treated with BM sera (50% diluted with serum free DMEM medium) from MM patients (n = 8) containing low (L; 2.7 to 8.5 ng/mL)

    Journal:

    Article Title: Myeloma-derived Dickkopf-1 disrupts Wnt-regulated osteoprotegerin and RANKL production by osteoblasts: a potential mechanism underlying osteolytic bone lesions in multiple myeloma

    doi: 10.1182/blood-2008-01-132134

    Figure Lengend Snippet: Neutralization of DKK1 rescued OPG expression in osteoblasts grown in the presence of MM sera or primary MM cells. C2C12 cells were treated with BM sera (50% diluted with serum free DMEM medium) from MM patients (n = 8) containing low (L; 2.7 to 8.5 ng/mL)

    Article Snippet: Mouse pluripotent mesenchymal precursor cell line C2C12 that has the potential of differentiating into osteoblast in the presence of BMP-2 and human osteoblast cell line hFOB1.19 were purchased from ATCC (Manassas, VA).

    Techniques: Neutralization, Expressing

    Wnt3a induced increase in OPG mRNA and protein in osteoblast progenitor cells. C2C12 cells (A,C) and Saos-2 cells (B,D) were treated with serial concentrations of recombinant Wnt3a for indicated times. The OPG mRNA (A,B) was amplified by qPCR analysis.

    Journal:

    Article Title: Myeloma-derived Dickkopf-1 disrupts Wnt-regulated osteoprotegerin and RANKL production by osteoblasts: a potential mechanism underlying osteolytic bone lesions in multiple myeloma

    doi: 10.1182/blood-2008-01-132134

    Figure Lengend Snippet: Wnt3a induced increase in OPG mRNA and protein in osteoblast progenitor cells. C2C12 cells (A,C) and Saos-2 cells (B,D) were treated with serial concentrations of recombinant Wnt3a for indicated times. The OPG mRNA (A,B) was amplified by qPCR analysis.

    Article Snippet: Mouse pluripotent mesenchymal precursor cell line C2C12 that has the potential of differentiating into osteoblast in the presence of BMP-2 and human osteoblast cell line hFOB1.19 were purchased from ATCC (Manassas, VA).

    Techniques: Recombinant, Amplification, Real-time Polymerase Chain Reaction