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: Heliyon

Article Title: Differentiation of cardiomyocyte-like cells from human amniotic fluid mesenchymal stem cells by combined induction with human platelet lysate and 5-azacytidine

doi: 10.1016/j.heliyon.2020.e04844

Figure Lengend Snippet: The primers for RT–qPCR and products size.

Article Snippet: After fixation, the cells were blocked in 10% AB-serum in 1% BSA-PBS for 30 min at room temperature, and then incubated with mouse anti-human Cx43 primary antibodies (Sigma-aldrich, USA) for 2 h at 37 °C.

Techniques:

Journal: Heliyon

Article Title: Differentiation of cardiomyocyte-like cells from human amniotic fluid mesenchymal stem cells by combined induction with human platelet lysate and 5-azacytidine

doi: 10.1016/j.heliyon.2020.e04844

Figure Lengend Snippet: A: hPL ability to up-regulate cardiomyogenic specific genes expression. Each graph displays the expression levels of GATA4 , cTnT , Cx4 3 and Nkx2.5 , which were normalized to GAPDH and were relative to the control group. Data are presented as mean ± S.E. values. ∗ statistically significant versus control. # statistically significant between group. B: hPL combined with 5-aza ability to up-regulate cardiomyogenic specific genes expression. Each graph displays the expression levels of GATA4 , cTnT , Cx43 and Nkx2.5 , which were normalized to GAPDH and were relative to the control group. Data are presented as mean ± S.E. values. ∗ statistically significant versus control. # statistically significant between group.

Article Snippet: After fixation, the cells were blocked in 10% AB-serum in 1% BSA-PBS for 30 min at room temperature, and then incubated with mouse anti-human Cx43 primary antibodies (Sigma-aldrich, USA) for 2 h at 37 °C.

Techniques: Expressing

Journal: Heliyon

Article Title: Differentiation of cardiomyocyte-like cells from human amniotic fluid mesenchymal stem cells by combined induction with human platelet lysate and 5-azacytidine

doi: 10.1016/j.heliyon.2020.e04844

Figure Lengend Snippet: Ability of hPL to promote the differentiation of hAF-MSCs into cardiomyocyte-like cells. Each graph displays the expression levels of GATA4 , cTnT , Cx43 and Nkx2. 5 that were normalized to GAPDH and were relative to the control group. Data are presented as mean ± S.E. values. ∗ statistically significant versus control. # statistically significant between group.

Article Snippet: After fixation, the cells were blocked in 10% AB-serum in 1% BSA-PBS for 30 min at room temperature, and then incubated with mouse anti-human Cx43 primary antibodies (Sigma-aldrich, USA) for 2 h at 37 °C.

Techniques: Expressing

Journal: Heliyon

Article Title: Differentiation of cardiomyocyte-like cells from human amniotic fluid mesenchymal stem cells by combined induction with human platelet lysate and 5-azacytidine

doi: 10.1016/j.heliyon.2020.e04844

Figure Lengend Snippet: Detection of cardiomyogenic specific proteins; immunofluorescence staining FITC (495 nm/519 nm), green color for all cardiomyogenic specific proteins (A–I) and immunoenzymatic staining (J–L); GATA4 (localized in nucleus) staining (A) control group, (B) 10 μM 5-aza induced group, (C) 10 μM 5-aza with 20% hPL induced group; cTnT (localized in cytoplasm) staining (D) control group, (E) 10 μM 5-aza induced group, (F) 10 μM 5-aza 20% hPL induced group; Nkx2.5 (localized in nucleus) staining (G) control group, (H) 10 μM 5-aza induced group, (I) 10 μM 5-aza with 20% hPL induced group; Cx43 (localized in cell membrane) staining (J) control group, (K) 10 μM 5-aza induced group (black arrow), (L) 10 μM 5-aza with 20% hPL induced group (black arrow). (A-C and G-I) insets without nuclear counterstain showing no nuclear staining of two key core cardiac transcription factors. Scale bar = 100 μm.

Article Snippet: After fixation, the cells were blocked in 10% AB-serum in 1% BSA-PBS for 30 min at room temperature, and then incubated with mouse anti-human Cx43 primary antibodies (Sigma-aldrich, USA) for 2 h at 37 °C.

Techniques: Immunofluorescence, Staining

Journal: Heliyon

Article Title: Differentiation of cardiomyocyte-like cells from human amniotic fluid mesenchymal stem cells by combined induction with human platelet lysate and 5-azacytidine

doi: 10.1016/j.heliyon.2020.e04844

Figure Lengend Snippet: Image J analysis showing the results of CTCF (the expression levels of GATA4, cTnT, Nkx2.5 and Cx43 proteins signal). Data are presented as mean ± S.E. values. ∗ statistically significant versus control.

Article Snippet: After fixation, the cells were blocked in 10% AB-serum in 1% BSA-PBS for 30 min at room temperature, and then incubated with mouse anti-human Cx43 primary antibodies (Sigma-aldrich, USA) for 2 h at 37 °C.

Techniques: Expressing