Journal: Journal of Cell Communication and Signaling

Article Title: CCN2 exerts direct cytoprotective actions in adult cardiac myocytes by activation of the PI3-kinase/Akt/GSK-3β signaling pathway

doi: 10.1007/s12079-012-0183-1

Figure Lengend Snippet: a . Western blots of indicated phospho-protein levels and total protein levels in cardiac myocytes stimulated with 100 nmol/L or 250 nmol/L rhCCN2 for 30 min. Figure demonstrates three replicate lanes of each treatment (control/stimulation). The Western blot images are all representative of three independent experiments. b . Densitometric analysis of Western blots in a . Histograms show phospho-protein levels relative to total protein contents. Data are mean ± SEM of n = 3 in each group, * p < 0.05 vs control

Article Snippet: Recombinant full-length, human CCN2 (rhCCN2) was contract-purified from the cell culture medium of transfected HEK 293 cells by EMP Genetech (Germany), essentially as previously described (Ahmed et al. ).

Techniques: Western Blot, Control

Journal: Journal of Cell Communication and Signaling

Article Title: CCN2 exerts direct cytoprotective actions in adult cardiac myocytes by activation of the PI3-kinase/Akt/GSK-3β signaling pathway

doi: 10.1007/s12079-012-0183-1

Figure Lengend Snippet: Activation of Akt/GSK-3β signaling pathway by CCN2. a Western blot analysis of phospho-Akt (Ser473) and b phospho-GSK-3β (Ser9) contents in cardiac myocytes stimulated with increasing concentrations of rhCCN2 for 30 min. c Western blot analysis of phospho-glycogen synthase (Ser641) levels in cardiac myocytes stimulated with 250 nmol/L rhCCN2 for 24 and 40 h. The histograms demonstrate densitometric analysis of phospho-protein levels relative to respective total protein contents. Data are mean ± SEM of n = 3 in each group, * p < 0.05 vs. control. The figure is representative of three independent experiments

Article Snippet: Recombinant full-length, human CCN2 (rhCCN2) was contract-purified from the cell culture medium of transfected HEK 293 cells by EMP Genetech (Germany), essentially as previously described (Ahmed et al. ).

Techniques: Activation Assay, Western Blot, Control

Journal: Journal of Cell Communication and Signaling

Article Title: CCN2 exerts direct cytoprotective actions in adult cardiac myocytes by activation of the PI3-kinase/Akt/GSK-3β signaling pathway

doi: 10.1007/s12079-012-0183-1

Figure Lengend Snippet: CCN2-mediated activation of Akt/GSK-3β signaling pathway is sensitive to PI3-kinase- and Akt-inhibiton. a Western blot analyses of phosphoprotein and total protein levels of Akt, b - c GSK-3β, and d p70S6K in cardiac myocytes incubated for 15 min without (Control) or with 100 nmol/L rhCCN2 (CCN2), or with 100 nmol/L rhCCN2 in the presence of the PI3-kinase inhibitor LY294002 (CCN2 + LY) or in presence of the Akt inhibitor API-2 (CCN2 + API-2). e Inhibition of phospho-Akt (Ser473) activities by competitive inhibition of pleckstrin homology (PH) domain of Akt by expression of GRK2ct. Western blot demonstrating phospho-Akt (Ser473) and total Akt levels in cardiac myocytes expressing GRK2ct or LacZ in the absence or presence of rhCCN2. Cells were incubated for 40 h after infection with Ad-GRK2ct or Ad-LacZ at 1,000× multiplicity of infection (MOI), then stimulated with rhCCN2 (100 nmol/L) for 30 min. f Western blot analysis of GRK2ct expression in cardiac myocytes 40 h after infection with 1,000 MOI Ad-GRK2ct or Ad-LacZ. The histograms demonstrate densitometric analysis of indicated phospho-protein activities relative to total contents of the respective proteins. Data are mean ± SEM, n = 3 in each group, * p < 0.05. The figure is representative of three independent experiments

Article Snippet: Recombinant full-length, human CCN2 (rhCCN2) was contract-purified from the cell culture medium of transfected HEK 293 cells by EMP Genetech (Germany), essentially as previously described (Ahmed et al. ).

Techniques: Activation Assay, Western Blot, Incubation, Control, Inhibition, Expressing, Infection

Journal: Journal of Cell Communication and Signaling

Article Title: CCN2 exerts direct cytoprotective actions in adult cardiac myocytes by activation of the PI3-kinase/Akt/GSK-3β signaling pathway

doi: 10.1007/s12079-012-0183-1

Figure Lengend Snippet: Comparison of efficacy of deglycosylated rhCCN2, heat-treated rhCCN2, and C-peptide with native rhCCN2. Concentration-effect curves of deglycosylated rhCCN2 ( a - b ), heat-treated ( c - d ), and C-peptide ( e - f ) compared with rhCCN2 in Rat-2–fibroblasts. The phosphoprotein levels were examined by Bio-Plex/Luminex. Each point is mean ± SEM ( n = 3)

Article Snippet: Recombinant full-length, human CCN2 (rhCCN2) was contract-purified from the cell culture medium of transfected HEK 293 cells by EMP Genetech (Germany), essentially as previously described (Ahmed et al. ).

Techniques: Comparison, Concentration Assay, Luminex

Journal: Journal of Cell Communication and Signaling

Article Title: CCN2 exerts direct cytoprotective actions in adult cardiac myocytes by activation of the PI3-kinase/Akt/GSK-3β signaling pathway

doi: 10.1007/s12079-012-0183-1

Figure Lengend Snippet: Assessment of integrity and homogeneity of rhCCN2. a Purified recombinant human CCN2 resolved by SDS-PAGE and stained with Coomassie brilliant blue (lane 1) or subjected to Western blot analysis (lane 2). b Coomassie staining of native rhCCN2 (Control) and deglycosylated rhCCN2 (Deglycos.)

Article Snippet: Recombinant full-length, human CCN2 (rhCCN2) was contract-purified from the cell culture medium of transfected HEK 293 cells by EMP Genetech (Germany), essentially as previously described (Ahmed et al. ).

Techniques: Purification, Recombinant, SDS Page, Staining, Western Blot, Control

Journal: Journal of Cell Communication and Signaling

Article Title: CCN2 exerts direct cytoprotective actions in adult cardiac myocytes by activation of the PI3-kinase/Akt/GSK-3β signaling pathway

doi: 10.1007/s12079-012-0183-1

Figure Lengend Snippet: a Phospho-Akt levels in Rat-2-fibroblasts stimulated in the absence or presence of rhCCN2 (50 nmol/L) for 30 min, or co-incubated with P1 and/or P2 in two different concentrations (12.5 μmol/L (μM) and 50 μmol/L) together with rhCCN2 (50 nmol/L). Phosphoprotein activities were analyzed by Bio-Plex/Luminex. Each point is mean ± SEM ( n = 3). * p < 0.05 vs. CCN2, † p < 0.05 vs. CCN2 +12.5 μmol/L P1 and vs. CCN2 +12.5 μmol/L P2, # p < 0.05 vs. CCN2 +50 μmol/L P1. b Diagram of the modules of CCN2, with the peptides derived from the Thrombospondin-homology (P1) and the IGFBP-homology (P2) domains marked

Article Snippet: Recombinant full-length, human CCN2 (rhCCN2) was contract-purified from the cell culture medium of transfected HEK 293 cells by EMP Genetech (Germany), essentially as previously described (Ahmed et al. ).

Techniques: Incubation, Luminex, Derivative Assay

Journal: Journal of Cell Communication and Signaling

Article Title: CCN2 exerts direct cytoprotective actions in adult cardiac myocytes by activation of the PI3-kinase/Akt/GSK-3β signaling pathway

doi: 10.1007/s12079-012-0183-1

Figure Lengend Snippet: Cardiac myocyte cell death quantified by analysis of release of lactate dehydrogenase (LDH) and adenylate kinase activities into the cell culture medium. a Viability of cardiac myocytes pretreated in the absence (Dox) or presence (Dox + CCN2) of 200 nmol/L rhCCN2 for 20 h, and then incubated with 15 μmol/L doxorubicin for 20 h. b Viability of cardiac myocytes preincubated in the absence (Control hypoxia) or presence (CCN2) of 200 nmol/L rhCCN2 for 20 h, then exposed to hypoxia (1 % O 2 ) for four hours, before one hour reoxygenation with ambient O 2 (simulated ischemia/reperfusion). Cell viability is expressed as the mean ± SEM percentage of the survival of untreated control cells (Control/Control normoxia), n = 6 in each group, * p < 0.05. The figure is representative of three independent experiments

Article Snippet: Recombinant full-length, human CCN2 (rhCCN2) was contract-purified from the cell culture medium of transfected HEK 293 cells by EMP Genetech (Germany), essentially as previously described (Ahmed et al. ).

Techniques: Cell Culture, Incubation, Control

Journal: Journal of Cell Communication and Signaling

Article Title: CCN2 exerts direct cytoprotective actions in adult cardiac myocytes by activation of the PI3-kinase/Akt/GSK-3β signaling pathway

doi: 10.1007/s12079-012-0183-1

Figure Lengend Snippet: Analysis of cell death quantified by LDH and adenylate kinase released into the cell culture medium of cardiac myocytes exposed to hypoxia (1 % O 2 ) for four hours and subsequently one hour reoxygenation to ambient O 2 (simulated ischemia/reperfusion). a - b Viability of cells preincubated for 20 h in the absence (Control hypoxia) or presence of 200 nmol/L rhCCN2 (CCN2), or with 200 nmol/L rhCCN2 and 10 μmol/L of PI3-kinase inhibitor LY294002 (CCN2 + LY294002), or 200 nmol/L rhCCN2 and 10 μmol/L LY303511 (Negative control with respect to PI3-kinase inhibitory activity of LY294002) (CCN2 + LY303511), before exposure to hypoxia and reoxygenation. Control cells were not exposed to hypoxia or rhCCN2 (Control normoxia). Cell viability is expressed as the mean ± SEM percentage of the survival of untreated control cells, n = 6 in each group, * p < 0.05 vs. control. c TUNEL of cells preincubated in the absence or presence of 200 nmol/L rhCCN2 for 20 h, then exposed to hypoxia and subsequently reoxygenation. Histogram demonstrates mean of three independent assays (number of % apoptotic cells from a minimum of 10 visual fields per assay). Data are mean ± SEM. * p < 0.05 vs. control. d Example of TUNEL positive cardiac myocytes. e Example of TUNEL negative cardiac myocytes

Article Snippet: Recombinant full-length, human CCN2 (rhCCN2) was contract-purified from the cell culture medium of transfected HEK 293 cells by EMP Genetech (Germany), essentially as previously described (Ahmed et al. ).

Techniques: Cell Culture, Control, Negative Control, Activity Assay, TUNEL Assay

Journal: Journal of Cell Communication and Signaling

Article Title: CCN2 exerts direct cytoprotective actions in adult cardiac myocytes by activation of the PI3-kinase/Akt/GSK-3β signaling pathway

doi: 10.1007/s12079-012-0183-1

Figure Lengend Snippet: Heat maps illustrating relative expression levels of differentially expressed genes relating to the Gene Ontology terms “response to wounding” and “anti-apoptosis” in cardiac myocytes stimulated in the absence or presence of rhCCN2 (200 nmol/L) for 48 h. Yellow/red and blue colors indicate expression levels above and below average, respectively

Article Snippet: Recombinant full-length, human CCN2 (rhCCN2) was contract-purified from the cell culture medium of transfected HEK 293 cells by EMP Genetech (Germany), essentially as previously described (Ahmed et al. ).

Techniques: Expressing

Journal: Journal of Cell Communication and Signaling

Article Title: CCN2 exerts direct cytoprotective actions in adult cardiac myocytes by activation of the PI3-kinase/Akt/GSK-3β signaling pathway

doi: 10.1007/s12079-012-0183-1

Figure Lengend Snippet: Heat maps illustrating relative expression levels of differentially expressed genes relating to the Gene Ontology terms “response to stress” and “apoptosis” (apoptosis-related genes) in cardiac myocytes stimulated in the absence or presence of rhCCN2 (200 nmol/L) for 48 h. Yellow/red and blue colors indicate expression levels above and below average, respectively

Article Snippet: Recombinant full-length, human CCN2 (rhCCN2) was contract-purified from the cell culture medium of transfected HEK 293 cells by EMP Genetech (Germany), essentially as previously described (Ahmed et al. ).

Techniques: Expressing

Journal: Journal of Cell Communication and Signaling

Article Title: CCN2 exerts direct cytoprotective actions in adult cardiac myocytes by activation of the PI3-kinase/Akt/GSK-3β signaling pathway

doi: 10.1007/s12079-012-0183-1

Figure Lengend Snippet: The most up- and down-regulated genes from the DNA microarray analysis of cardiac myocytes cultured in the absence ( n = 6) or presence ( n = 6) of 200 nmol/L CCN2 for 48 h

Article Snippet: Recombinant full-length, human CCN2 (rhCCN2) was contract-purified from the cell culture medium of transfected HEK 293 cells by EMP Genetech (Germany), essentially as previously described (Ahmed et al. ).

Techniques: Microarray, Cell Culture

Journal: Journal of Cell Communication and Signaling

Article Title: CCN2 exerts direct cytoprotective actions in adult cardiac myocytes by activation of the PI3-kinase/Akt/GSK-3β signaling pathway

doi: 10.1007/s12079-012-0183-1

Figure Lengend Snippet: Histograms of quantitative real-time RT-PCR analyses of mRNA levels of WISP1, GDF15, Lipocalin-2, and DYRK3, relative to 18S rRNA levels in cardiac myocytes incubated for 48 h without rhCCN2, with 200 nmol/L rhCCN2, or with 200 nmol/L rhCCN2 in combination with 10 μmol/L API-2. Data are mean ± SEM, n = 3 in each group, * p < 0.05

Article Snippet: Recombinant full-length, human CCN2 (rhCCN2) was contract-purified from the cell culture medium of transfected HEK 293 cells by EMP Genetech (Germany), essentially as previously described (Ahmed et al. ).

Techniques: Quantitative RT-PCR, Incubation