Journal: International Journal of Molecular Sciences

Article Title: Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

doi: 10.3390/ijms23010294

Figure Lengend Snippet: Downregulation of Cx43 reduces endothelial cell migration. ( a ) Western blot analysis of Cx43, Cx40, and Cx37 revealed that Cx43 is expressed in both human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC), whereas Cx40 and Cx37 are not expressed in HMEC. Cx43 (Cx43), Cx40 (Cx), and Cx37 (Cx) expressing HeLa cells were used as the positive controls. CTL: Wildtype HeLa cell lysate. ( b ) Western blot analysis of Cx43 in HMEC transfected with 100 nM siRNA against Cx43 or non-silencing control siRNA (Ctrl) for 48 h. The Cx43 expression was analysed using a polyclonal Cx43 antibody. Immunostaining for GAPDH was used as the loading control. ( c ) Serum-induced cell migration of HMEC transfected with a Cx43-siRNA or a control siRNA (Ctrl) in response to 10% FCS was assessed in a wound assay. Downregulation of Cx43 significantly decreased cell migration, represented as accumulated distance (*** p < 0.001 Ctrl-siRNA vs. Cx43-siRNA, n = 6, 3 independent cell cultures). ( d ) Representative single cell traces of migrated HMEC.

Article Snippet: For immunoprecipitation, the supernatants were incubated over night at 4 °C with a mouse anti-SHP-2 antibody (Santa Cruz) or a rabbit anti-Cx43 antibody (Sigma Aldrich), and were magnetic beads coated with protein A (Cx43) or G (SHP-2) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Migration, Western Blot, Expressing, Transfection, Control, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

doi: 10.3390/ijms23010294

Figure Lengend Snippet: Downregulation of Cx43 impairs vessel sprouting ex vivo. ( a ) Downregulation of Cx43 in the mouse aortae was achieved by transfection with Cx43 siRNA (500 nM) or control (Ctrl) siRNA, as assessed by Western blot 48 h later. ( b ) Representative images of aortic sprouts in matrigel. Scale bar represents 200 µm. ( c ) The vessel sprouting area (** p < 0.01 Ctrl-siRNA vs. Cx43-siRNA, n = 2 aortae with eight aortic segments each at 3 days; n = 3–4 aortae with 6–8 aortic segments each at 6 days) and ( d ) the number of sprout bifurcations (** p < 0.01 Ctrl-siRNA vs. Cx43-siRNA, n = 2 aortae with 8 aortic segments each at 3 days; *** p < 0.001 Ctrl-siRNA vs. Cx43-siRNA n = 3–4 aortae with 6–8 aortic segments each at 6 days) were significantly reduced upon downregulation of Cx43, as assessed 3 days and 6 days after transfection in a matrigel assay.

Article Snippet: For immunoprecipitation, the supernatants were incubated over night at 4 °C with a mouse anti-SHP-2 antibody (Santa Cruz) or a rabbit anti-Cx43 antibody (Sigma Aldrich), and were magnetic beads coated with protein A (Cx43) or G (SHP-2) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Ex Vivo, Transfection, Control, Western Blot, Matrigel Assay

Journal: International Journal of Molecular Sciences

Article Title: Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

doi: 10.3390/ijms23010294

Figure Lengend Snippet: Interaction of Cx43 and SHP-2 induces SHP-2 phosphatase activity. ( a ) Upon immunoprecipitation (IP) of SHP-2 from HeLa cells expressing only Cx43, Cx43 was co-immunoprecipitated. As a control, cells transfected with an empty vector (control (CTL)) were used. L: Whole cell lysates. ( b ) SHP-2 was detected upon immunoblotting after immunoprecipitation of Cx43 from HeLa cells expressing only Cx43 in contrast to the control cells (CTL). L: Whole cell lysates. Images originates from the same blot, which was cropped; ( c ) SHP-2 was immunoprecipitated together with Cx43 in HMEC; ( d ) Co-localization of SHP-2 and Cx43 was detected by immunofluorescent staining of HMEC followed by confocal microscopic imaging. Scale bar represents 20 µm; ( e ) SHP-2 phosphatase activity was significantly increased in HeLa cells expressing Cx43 compared to HeLa cells transfected with an empty vector (* p < 0.05, n = 4 independent cell cultures), as assessed by detection of dephosphorylation of a phosphate analogue in SHP-2 immunoprecipitates.

Article Snippet: For immunoprecipitation, the supernatants were incubated over night at 4 °C with a mouse anti-SHP-2 antibody (Santa Cruz) or a rabbit anti-Cx43 antibody (Sigma Aldrich), and were magnetic beads coated with protein A (Cx43) or G (SHP-2) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Activity Assay, Immunoprecipitation, Expressing, Control, Transfection, Plasmid Preparation, Western Blot, Staining, Imaging, De-Phosphorylation Assay

Journal: International Journal of Molecular Sciences

Article Title: Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

doi: 10.3390/ijms23010294

Figure Lengend Snippet: Inactivation of SHP-2 reduces endothelial cell migration. ( a ) Overexpression of a dominant negative substrate trapping mutant SHP-2 (SHP-2 CS) in HMEC significantly reduced serum (10%) induced endothelial cell migration, as assessed by the accumulated distance in µm in an in vitro wound assay (* p < 0.05, n = 3 independent cell cultures) compared to SHP-2 WT. Additional knock-down of Cx43 (Cx43 siRNA) did not further decrease migration (ns: not significant, n = 3 independent cell cultures). ( b ) Representative single cell traces of migrated HMEC.

Article Snippet: For immunoprecipitation, the supernatants were incubated over night at 4 °C with a mouse anti-SHP-2 antibody (Santa Cruz) or a rabbit anti-Cx43 antibody (Sigma Aldrich), and were magnetic beads coated with protein A (Cx43) or G (SHP-2) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Migration, Over Expression, Dominant Negative Mutation, Mutagenesis, In Vitro, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

doi: 10.3390/ijms23010294

Figure Lengend Snippet: The Cx43 and SHP-2 interaction is vital for endothelial migration. ( a ) Treatment of HMEC overexpressing a constitutively active mutant SHP-2 (SHP-2 EA) with Cx43 siRNA did not rescue the reduced migratory response caused by Cx43 knock-down (Cx43 siRNA) (ns: not significant, n = 3 independent cell cultures) compared to SHP-2 EA cells treated with control siRNA (Ctrl) (* p < 0.05, n = 3 independent cell cultures), as assessed by the accumulated distance in µm in an in vitro wound assay (* p < 0.05, n = 3 independent cell cultures). ( b ) Representative single cell traces of migrated HMEC.

Article Snippet: For immunoprecipitation, the supernatants were incubated over night at 4 °C with a mouse anti-SHP-2 antibody (Santa Cruz) or a rabbit anti-Cx43 antibody (Sigma Aldrich), and were magnetic beads coated with protein A (Cx43) or G (SHP-2) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Migration, Mutagenesis, Knockdown, Control, In Vitro

Journal: PLoS ONE

Article Title: Induction and Enhancement of Cardiac Cell Differentiation from Mouse and Human Induced Pluripotent Stem Cells with Cyclosporin-A

doi: 10.1371/journal.pone.0016734

Figure Lengend Snippet: A. Gross appearance of cardiomyocyte induction by CSA. Six days after the Flk1 + cell culture on OP9 cells (Flk-d6). cTnT staining (brown). Left panel: control. Right panel: CSA treatment. Scale bars = 400 µm. B. Quantitative evaluation of cardiomyocyte induction by fluorescent intensity of cTnT staining. Relative fluorescent intensity is indicated (n = 4, †, p<0.001 vs control). C. Representative action potential of iPSC-derived spontaneously beating cardiomyocytes. D. Sarcomeric organization in TMRM-purified cardiomyocytes at Flk-d8. Immunostaining with anti-sarcomeric α-actinin antibody (red) and DAPI (blue). Right panel shows higher magnification of boxed area. Scale bar = 25 µm. E–H. Double immunostaining of TMRM-purified cardiomyocytes at Flk-d8 for connexin43 (Cx43) (green) and cTnT (orange) (E), Cav3.2 (green) and cTnT (orange) (F), HCN4 (green) and cTnT (orange) (G), Kir2.1 (green) and cTnT (orange) (H). Nuclei are visualized with DAPI. Scale bars = 25 µm. I. FACS analysis for cardiac progenitor induction from mouse iPSCs by CSA. X axis: CXCR4. Y axis: Flk1. Percentages of FCV cardiac progenitor cells (double positive population; red boxes) in total Flk1 + cell progenies are indicated.

Article Snippet: Anti-Kir2.1 (1∶200) and anti-connexin 43 (1∶200) antibodies were from Alomone (Israel) and Invitrogen (Carlsbad, CA), respectively.

Techniques: Cell Culture, Staining, Derivative Assay, Purification, Immunostaining, Double Immunostaining

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: The Dome Wall of Bladder Acts as a Pacemaker Site in Detrusor Instability in Rats

doi: 10.12659/MSM.904406

Figure Lengend Snippet: List of primers used in PCR.

Article Snippet: Tissues sections were incubated with primary antibody dilutions made up in 0.2% BSA/0.01 MPBS: goat anti-mouse c-KIT (1: 100, Santa Cruz, Germany), rabbit anti-mouse HCN2 (1: 100, Chemicon, Japan), and rabbit anti-mouse Cx43 (1: 100, Cell Signaling, USA) overnight at 4°C, respectively.

Techniques: Sequencing

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: The Dome Wall of Bladder Acts as a Pacemaker Site in Detrusor Instability in Rats

doi: 10.12659/MSM.904406

Figure Lengend Snippet: The expression of Connexin43. ( A ) Connexin43 mRNAs and ( B ) protein expression in DI group. ( C ) Co-expression of Connexin-43 and c-kit-positive ICCs in the DI detrusor. * P <0.05 vs. other parts in DI group.

Article Snippet: Tissues sections were incubated with primary antibody dilutions made up in 0.2% BSA/0.01 MPBS: goat anti-mouse c-KIT (1: 100, Santa Cruz, Germany), rabbit anti-mouse HCN2 (1: 100, Chemicon, Japan), and rabbit anti-mouse Cx43 (1: 100, Cell Signaling, USA) overnight at 4°C, respectively.

Techniques: Expressing

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: The Dome Wall of Bladder Acts as a Pacemaker Site in Detrusor Instability in Rats

doi: 10.12659/MSM.904406

Figure Lengend Snippet: The expression of Connexin43. ( A ) HCN2 mRNAs and ( B ) protein expression in DI group. ( C ) Co-expression of HCN2 and c-kit-positive ICCs in the DI detrusor. * P <0.05 vs. other parts in DI group.

Article Snippet: Tissues sections were incubated with primary antibody dilutions made up in 0.2% BSA/0.01 MPBS: goat anti-mouse c-KIT (1: 100, Santa Cruz, Germany), rabbit anti-mouse HCN2 (1: 100, Chemicon, Japan), and rabbit anti-mouse Cx43 (1: 100, Cell Signaling, USA) overnight at 4°C, respectively.

Techniques: Expressing

Journal:

Article Title: Connexin Expression in Renin-Producing Cells

doi: 10.1681/ASN.2008030252

Figure Lengend Snippet: (A through D) Histologic sections of kidney cortex from a mouse kept on normal-salt diet stained for Cx40 (A), Cx37 (B), Cx43 (C), Cx45 (D) together with renin (green color). Bar = 50 μm. Circles highlight glomerulus (gl); egm, extraglomerular mesangium; aa, afferent arteriole; arrowheads highlight renin-expressing cells; arrows indicate endothelial Cx staining; ▵, Cx45 staining in smooth muscle cells. Cx37, Cx40, and Cx43 are found in association with renin-expressing cells and with endothelial cells of the aa. Whereas Cx40 and Cx43 immunoreactivity extends more evenly over the whole glomeruli, Cx37 immunoreactivity is more focused to the entrance of the aa into the capillary network. Cx45 immunoreactivity is not associated with renin-expressing cells but clearly is seen in VSMCs of aa and in intraglomerular cells.

Article Snippet: Antibodies used for immunostaining of Cxs were purchased from commercial providers: Anti-Cx40 (goat anti-mouse; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Cx37 (rabbit anti-mouse; Alpha Diagnostic Int., San Antonio, TX), anti-Cx43 (rabbit anti-mouse; Sigma, St. Louis, MO, and Zymed, Basel, Switzerland), anti-Cx45 (rabbit anti-mouse 32 ), and anti–α-SMA (mouse anti-mouse; Beckman Coulter Immunotech, Marseille, France).

Techniques: Staining, Expressing

Journal:

Article Title: Connexin Expression in Renin-Producing Cells

doi: 10.1681/ASN.2008030252

Figure Lengend Snippet: (A through D) Histologic sections of kidney cortex from a mouse kept on low-salt diet in combination with the ACEI enalapril (10 mg/kg per d), stained for Cx40 (A), Cx37 (B), Cx43 (C), and Cx45 (D) together with renin (green color). Bar = 50 μm. Circles highlight gl; arrowheads highlight renin-expressing cells; arrows indicate renin immunoreactivity without immunoreactivity of the respective Cx. Treatment of the mice led to an upstream recruitment of renin-expressing cells along the aa. All renin positive cells also stained for Cx40, whereas Cx37 immunoreactivity was absent from renin-expressing cells but still present in the endothelium of aa. Cx43 immunoreactivity was associated with renin-expressing cells close to the glomerular vascular poles but faded in renin-expressing cells with increasing distance to the vascular pole. Cx45 immunoreactivity did not co-localize with renin.

Article Snippet: Antibodies used for immunostaining of Cxs were purchased from commercial providers: Anti-Cx40 (goat anti-mouse; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Cx37 (rabbit anti-mouse; Alpha Diagnostic Int., San Antonio, TX), anti-Cx43 (rabbit anti-mouse; Sigma, St. Louis, MO, and Zymed, Basel, Switzerland), anti-Cx45 (rabbit anti-mouse 32 ), and anti–α-SMA (mouse anti-mouse; Beckman Coulter Immunotech, Marseille, France).

Techniques: Staining, Expressing

Journal:

Article Title: Connexin Expression in Renin-Producing Cells

doi: 10.1681/ASN.2008030252

Figure Lengend Snippet: Renin, Cx37, Cx40, Cx43, and Cx45 mRNA abundance in kidney cortex of mice treated with a combination of low-salt diet (0.02% wt/wt) with the ACEI enalapril (10 mg/kg per d; ls/enal), with enalapril (10 mg/kg per d) alone (enal), with low-salt diet (0.02% wt/wt; ls), with normal-salt diet (0.6% wt/wt), or with high-salt diet (4% wt/wt; hs). Data are means ± SEM of five mice in each group. *P < 0.05 versus vehicle-treated mice on normal-salt diet.

Article Snippet: Antibodies used for immunostaining of Cxs were purchased from commercial providers: Anti-Cx40 (goat anti-mouse; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Cx37 (rabbit anti-mouse; Alpha Diagnostic Int., San Antonio, TX), anti-Cx43 (rabbit anti-mouse; Sigma, St. Louis, MO, and Zymed, Basel, Switzerland), anti-Cx45 (rabbit anti-mouse 32 ), and anti–α-SMA (mouse anti-mouse; Beckman Coulter Immunotech, Marseille, France).

Techniques:

Journal:

Article Title: Connexin Expression in Renin-Producing Cells

doi: 10.1681/ASN.2008030252

Figure Lengend Snippet: Histologic sections of kidney cortex from a normal 17-d-old mouse fetus. (A) Immunostaining for Cx43 alone. (B) Co-immunostaining for renin (green) and Cx43 (red). (C) Co-immunostaining for renin (green), α-SMA (blue), and Cx43 (red). (D) Immunostaining for Cx45 alone. (E) Co-immunostaining for renin (green) and Cx45 (red). (F) Co-immunostaining for renin (green), α-SMA (blue), and Cx45 (red). Bar = 50 μm. Arrowheads indicate regions of renin expression without Cx staining; arrows indicate endothelial Cx staining; lines, Cx45 co-localizing with renin; *, Cx45 in smooth muscle cells with low Cx45 staining.

Article Snippet: Antibodies used for immunostaining of Cxs were purchased from commercial providers: Anti-Cx40 (goat anti-mouse; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Cx37 (rabbit anti-mouse; Alpha Diagnostic Int., San Antonio, TX), anti-Cx43 (rabbit anti-mouse; Sigma, St. Louis, MO, and Zymed, Basel, Switzerland), anti-Cx45 (rabbit anti-mouse 32 ), and anti–α-SMA (mouse anti-mouse; Beckman Coulter Immunotech, Marseille, France).

Techniques: Immunostaining, Expressing, Staining

Journal:

Article Title: Connexin Expression in Renin-Producing Cells

doi: 10.1681/ASN.2008030252

Figure Lengend Snippet: Histologic sections of kidney cortex from a Cx40-deficient mouse kept on a normal-salt diet. (A) Immunostaining for Cx40 alone. (B) Co-immunostaining for renin (green), Cx40 (red), and α-SMA (blue). (C) Immunostaining for Cx37 alone. (D) Co-immunostaining for renin (green), Cx37 (red), and α-SMA (blue). (E) Immunostaining for Cx43 alone. (F) Co-immunostaining for renin (green), Cx43 (red), and α-SMA (blue). Bar = 50 μm. Circles highlight gl; arrowheads indicate renin-expressing cells; arrows indicate endothelial Cx staining; ▵, staining for Cx43 in venous endothelium.

Article Snippet: Antibodies used for immunostaining of Cxs were purchased from commercial providers: Anti-Cx40 (goat anti-mouse; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Cx37 (rabbit anti-mouse; Alpha Diagnostic Int., San Antonio, TX), anti-Cx43 (rabbit anti-mouse; Sigma, St. Louis, MO, and Zymed, Basel, Switzerland), anti-Cx45 (rabbit anti-mouse 32 ), and anti–α-SMA (mouse anti-mouse; Beckman Coulter Immunotech, Marseille, France).

Techniques: Immunostaining, Expressing, Staining

Journal:

Article Title: Connexin Expression in Renin-Producing Cells

doi: 10.1681/ASN.2008030252

Figure Lengend Snippet: Primer sequences used for cDNA amplification

Article Snippet: Antibodies used for immunostaining of Cxs were purchased from commercial providers: Anti-Cx40 (goat anti-mouse; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Cx37 (rabbit anti-mouse; Alpha Diagnostic Int., San Antonio, TX), anti-Cx43 (rabbit anti-mouse; Sigma, St. Louis, MO, and Zymed, Basel, Switzerland), anti-Cx45 (rabbit anti-mouse 32 ), and anti–α-SMA (mouse anti-mouse; Beckman Coulter Immunotech, Marseille, France).

Techniques: Sequencing