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: Cell Communication and Signaling : CCS

Article Title: Hypoxia-preconditioning human bone marrow-derived mesenchymal stem cells induce high-quality mitochondrial transfer through gap junctions to alleviate ischemia-reperfusion injury in liver graft

doi: 10.1186/s12964-025-02497-1

Figure Lengend Snippet: Hypo-hBMSCs enhance GJs function via up-regulating Cx43 and Cx32 expression. A Public databases indicated the protein expression levels of the Cx in hBMSCs and liver tissues. B Western blotting showed levels of Cx43, Cx32 and Cx26 protein expression in hBMSCs and BRL3A cells. GAPDH served as a loading control. C RT-qPCR and Western blotting were used to detect Cx43, Cx32 and Cx26 mRNAs and proteins levels in hBMSCs/hypo-hBMSCs. β-actin served as a loading control. D Co-IP assay was used to determine whether heterotypic channels were formed. β-actin served as a loading control. Data are shown as mean ± SEM, n = 3–5. * p < 0.05, ** p < 0.01, *** p < 0.001. HBMSCs, human bone marrow mesenchymal stem cells; hypo-hBMSCs, hypoxia preconditioning-hBMSCs

Article Snippet: To establish a HR cell model, BRL3A cells with or without hBMSCs were processed using the same protocol as previously described for the hBMSCs, and placed in a Galaxy 48R hypoxia incubator (Eppendorf GmbH, Hamburg, Germany) with 5% CO 2 and 1% O 2 at 37 °C for 12 h. Subsequently, the cells were placed in the normal cell incubator for 2 h. To plumb the role of Cx43 and Cx32 in this study, retinoic acid (RA, 1 μM, MedChemExpress, New Jersey, USA), the specific GJs agonist, and Gap26 (0.25 mg/mL, APExBIO, Houston, USA), the specific GJs inhibitor, were used in hBMSCs.

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Co-Immunoprecipitation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Hypoxia-preconditioning human bone marrow-derived mesenchymal stem cells induce high-quality mitochondrial transfer through gap junctions to alleviate ischemia-reperfusion injury in liver graft

doi: 10.1186/s12964-025-02497-1

Figure Lengend Snippet: Functional homotypic Cx43-GJs and Cx32-GJs mediate mitochondrial transfer from hypo-hBMSCs to hepatocytes. A Western blotting showed levels of Cx43, Cx32 in hBMSCs. GAPDH and β-actin served as a loading control. B - E Evaluation of GJs function and mitochondrial transfer. (B, D) “Parachute” dye-coupling assay measured the function of GJs. (C, E) Immunofluorescence staining showed the levels of mitochondrial transfer from hBMSCs or hypo-hBMSCs to BRL3A cells. Data are shown as mean ± SEM, n = 3–5. * p < 0.05, ** p < 0.01, *** p < 0.001. HBMSCs, human bone marrow mesenchymal stem cells; hypo-hBMSCs, hypoxia preconditioning-hBMSCs

Article Snippet: To establish a HR cell model, BRL3A cells with or without hBMSCs were processed using the same protocol as previously described for the hBMSCs, and placed in a Galaxy 48R hypoxia incubator (Eppendorf GmbH, Hamburg, Germany) with 5% CO 2 and 1% O 2 at 37 °C for 12 h. Subsequently, the cells were placed in the normal cell incubator for 2 h. To plumb the role of Cx43 and Cx32 in this study, retinoic acid (RA, 1 μM, MedChemExpress, New Jersey, USA), the specific GJs agonist, and Gap26 (0.25 mg/mL, APExBIO, Houston, USA), the specific GJs inhibitor, were used in hBMSCs.

Techniques: Functional Assay, Western Blot, Control, Immunofluorescence, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Hypoxia-preconditioning human bone marrow-derived mesenchymal stem cells induce high-quality mitochondrial transfer through gap junctions to alleviate ischemia-reperfusion injury in liver graft

doi: 10.1186/s12964-025-02497-1

Figure Lengend Snippet: Functional homotypic Cx43-GJs and Cx32-GJs mediate mitochondrial transfer from hypo-hBMSCs to hepatocytes. A Western blotting showed levels of Cx43, Cx32 in hBMSCs. B “Parachute” dye-coupling assay measured the function of GJs. C Immunofluorescence staining showed the levels of mitochondrial transfer from hBMSCs or hypo-hBMSCs to BRL3A cells. Data are shown as mean ± SEM, n = 3–5. * p < 0.05, ** p < 0.01, *** p < 0.001. HBMSCs, human bone marrow mesenchymal stem cells; hypo-hBMSCs, hypoxia preconditioning-hBMSCs

Article Snippet: To establish a HR cell model, BRL3A cells with or without hBMSCs were processed using the same protocol as previously described for the hBMSCs, and placed in a Galaxy 48R hypoxia incubator (Eppendorf GmbH, Hamburg, Germany) with 5% CO 2 and 1% O 2 at 37 °C for 12 h. Subsequently, the cells were placed in the normal cell incubator for 2 h. To plumb the role of Cx43 and Cx32 in this study, retinoic acid (RA, 1 μM, MedChemExpress, New Jersey, USA), the specific GJs agonist, and Gap26 (0.25 mg/mL, APExBIO, Houston, USA), the specific GJs inhibitor, were used in hBMSCs.

Techniques: Functional Assay, Western Blot, Immunofluorescence, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Hypoxia-preconditioning human bone marrow-derived mesenchymal stem cells induce high-quality mitochondrial transfer through gap junctions to alleviate ischemia-reperfusion injury in liver graft

doi: 10.1186/s12964-025-02497-1

Figure Lengend Snippet: Graphical scheme of Hypo-hBMSCs induce high-quality mitochondrial transfer through GJs to alleviate hepatic IRI. ① Hypo-hBMSCs initiated mitophagy to improve mitochondrial quality. ② Hypo-hBMSCs enhanced the expression of Cx43 and Cx32. ③ Hypo-hBMSCs facilitated efficient mitochondrial transfer to hepatocytes by enhancing Cx43-GJs and Cx32-GJs function. ④ Hypo-hBMSCs transferred a greater quantity and higher quality of mitochondria into hepatocytes, enabling the hepatocytes to produce more ATP and thereby alleviating Hepatic IRI. IRI, ischemia and reperfusion injury; HR, hypoxia/reoxygenation; hBMSCs, human bone marrow mesenchymal stem cells; hypo-hBMSCs, hypoxia preconditioning-hBMSC

Article Snippet: To establish a HR cell model, BRL3A cells with or without hBMSCs were processed using the same protocol as previously described for the hBMSCs, and placed in a Galaxy 48R hypoxia incubator (Eppendorf GmbH, Hamburg, Germany) with 5% CO 2 and 1% O 2 at 37 °C for 12 h. Subsequently, the cells were placed in the normal cell incubator for 2 h. To plumb the role of Cx43 and Cx32 in this study, retinoic acid (RA, 1 μM, MedChemExpress, New Jersey, USA), the specific GJs agonist, and Gap26 (0.25 mg/mL, APExBIO, Houston, USA), the specific GJs inhibitor, were used in hBMSCs.

Techniques: Expressing