dkk1 expression plasmid  (Agilent technologies)

Journal: PLoS ONE

Article Title: Negative Regulation of Bone Formation by the Transmembrane Wnt Antagonist Kremen-2

doi: 10.1371/journal.pone.0010309

Figure Lengend Snippet: (A) RT-PCR expression analysis of Dkk , Krm and Rspo genes in primary osteoblasts (Obl. d5, non-mineralized, Obl. d25, mineralized) and various tissues of 6 weeks old mice. (B) RT-PCR expression analysis of Krm genes in non-differentiated MC3T3-E1 cells and tissues of newborn mice. (C) Immunohistochemistry on human bone sections reveals that KRM2 is present on osteoblasts lining the trabecular bone surface (arrows, scale bars, 100 µm). The bottom panel shows staining of osteoclasts (scale bars, 20 µm). (D) DNA transfection in MC3T3-E1 cells using expression plasmids for Wnt1, Wnt2 or Wnt3, Dkk1 and/or Krm2 at the indicated combinations. Bars represent mean ± SD of three independent experiments (n = 9). Asterisks indicate statistically significant changes.

Article Snippet: The three different Wnt expression plasmids were kindly provided by Dr. J. Kitajewski (New York, USA), the Krm2 expression plasmid has been described previously , and the Dkk1 expression plasmid was constructed by placing the full-length cDNA into the vector pCMV-Tag4 (Stratagene).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemistry, Staining, Transfection

Journal: PLoS ONE

Article Title: Negative Regulation of Bone Formation by the Transmembrane Wnt Antagonist Kremen-2

doi: 10.1371/journal.pone.0010309

Figure Lengend Snippet: (A) Affymetrix Gene Chip hybridization demonstrates that several well-established osteoblast differentiation markers are expressed at similar levels in osteoblasts from wildtype (mean values indicated by the dotted red line) and transgenic mice, while other genes are expressed at lower levels in the latter ones. Bars represent mean ± SD (n = 3). Asterisks indicate statistically significant differences between the relative signal intensities in wildtype and transgenic samples. (B) Affymetrix Gene Chip hybridization of wildtype osteoblasts following treatment with Dkk1 for 6 hours at day 10 of differentiation (n = 1). Shown are the Affymetrix signal intensities and the signal log ratios (SLR) for the 25 genes displaying the strongest negative regulation by Dkk1. The mean signal ratios of the Gene Chip comparison between wildtype and Col1a1-Krm2 transgenic osteoblasts are given on the right.

Article Snippet: The three different Wnt expression plasmids were kindly provided by Dr. J. Kitajewski (New York, USA), the Krm2 expression plasmid has been described previously , and the Dkk1 expression plasmid was constructed by placing the full-length cDNA into the vector pCMV-Tag4 (Stratagene).

Techniques: Hybridization, Transgenic Assay

Journal: BMC immunology

Article Title: Wnt/β-catenin signaling reduces Bacillus Calmette-Guerin-induced macrophage necrosis through a ROS -mediated PARP/AIF-dependent pathway.

doi: 10.1186/s12865-015-0080-5

Figure Lengend Snippet: Figure 5 Activation of Wnt/β-catenin signaling increases GSH concentration in RAW264.7 cells. RAW264.7 cells were treated with BCG, Wnt3a, DKK1, H2O2 or their combination as indicated, they were then used for determination of intracellular GSH levels by an ELISA for GSH. (A) Impact of Wnt/β-catenin signaling on GSH production of RAW264.7 cells infected with BCG at MOI of 10 for 6 h. An activation of Wnt signaling by addition of Wnt3a-CM could induce GSH generation; in contrast, overexpression of Wnt inhibitor DKK1 reduced GSH levels. (B) Impact of Wnt/ β-catenin signaling on GSH production of RAW264.7 cells exposed to oxidative stress H2O2 (500 μmol/L) or LPS (100 ng/mL) stimulation for 6 h. The Wnt signaling could increase intracellular reduced GSH concentration in cells stressed by H2O2 and LPS. Compared to a control-CM treated cells, *: p < 0.05, **: p < 0.01. Control-CM treated cells compared to its corresponding Wnt3a-CM treated cells, #: p < 0.05; ##: p < 0.01. Data represented the mean ± SD from three independent triplicate experiments (N = 9).

Article Snippet: The plasmid expressing Wnt inhibitor DKK1 pCS2hDKK1-flag (Cat. #15494) was purchased from Addgene (www.addgene.com).

Techniques: Activation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Infection, Over Expression, Control

Journal: BMC Immunology

Article Title: Wnt/β-Catenin signaling reduces Bacillus Calmette-Guerin -induced macrophage necrosis through a ROS -mediated PARP/AIF-dependent pathway

doi: 10.1186/s12865-015-0080-5

Figure Lengend Snippet: Activation of Wnt/β-catenin signaling increases GSH concentration in RAW264.7 cells. RAW264.7 cells were treated with BCG, Wnt3a, DKK1, H 2 O 2 or their combination as indicated, they were then used for determination of intracellular GSH levels by an ELISA for GSH. (A) Impact of Wnt/β-catenin signaling on GSH production of RAW264.7 cells infected with BCG at MOI of 10 for 6 h. An activation of Wnt signaling by addition of Wnt3a-CM could induce GSH generation; in contrast, overexpression of Wnt inhibitor DKK1 reduced GSH levels. (B) Impact of Wnt/β-catenin signaling on GSH production of RAW264.7 cells exposed to oxidative stress H 2 O 2 (500 μmol/L) or LPS (100 ng/mL) stimulation for 6 h. The Wnt signaling could increase intracellular reduced GSH concentration in cells stressed by H 2 O 2 and LPS. Compared to a control-CM treated cells, *: p < 0.05, **: p < 0.01. Control-CM treated cells compared to its corresponding Wnt3a-CM treated cells, #: p < 0.05; ##: p < 0.01. Data represented the mean ± SD from three independent triplicate experiments (N = 9).

Article Snippet: The plasmid expressing Wnt inhibitor DKK1 pCS2-hDKK1-flag (Cat. #15494) was purchased from Addgene ( www.addgene.com ).

Techniques: Activation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Infection, Over Expression, Control