Journal: Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging

Article Title: In Situ Labeling and Magnetic Resonance Imaging of transplanted Human Hepatic Stem Cells

doi: 10.1007/s11307-010-0422-x

Figure Lengend Snippet: Schematics demonstrating the principle of contrast labeling cells for MR imaging. (a) Schematics of the magnetic microbeads coupled to an antibody. (b) Schematic of microbeads bound to a human hepatic stem cell, hHpSC. (c) Schematic to explain the enhancement of MRI by use of the microbeads.

Article Snippet: In this study, HEA-125 antibodies to EpCAM coupled to magnetic iron microbeads were obtained from Miltenyi Biotech (Bergisch Gladbach, Germany, recently cataloged as CD326 Microbead).

Techniques: Labeling, Imaging

Journal: Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging

Article Title: In Situ Labeling and Magnetic Resonance Imaging of transplanted Human Hepatic Stem Cells

doi: 10.1007/s11307-010-0422-x

Figure Lengend Snippet: Transmission Electron Microscopy (TEM, a–d), Scanning Electron Microscopy (SEM, e–i), and Emission Dispersive X-ray (EDX, Line graph and j) displaying human cells labeled with HEA-125 antibodies coupled to magnetic microbeads (Miltenyi). (a–d) Ability to detect the microbeads, and to distinguish them from glycogen particles (small arrows) occurs with aggregates yielding a size above 200 μm (large arrows); (e) SEM of hHpSCs that is not labeled with the beads; (f-iI) SEM of hHpSCs labeled with the beads (arrows denote the beads); (j) The line graph is from EDX analyses and shows that the beads on the cells contain the elements known to be in the EpCAM-microbeads, notably iron.

Article Snippet: In this study, HEA-125 antibodies to EpCAM coupled to magnetic iron microbeads were obtained from Miltenyi Biotech (Bergisch Gladbach, Germany, recently cataloged as CD326 Microbead).

Techniques: Transmission Assay, Electron Microscopy, Labeling

Journal: Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging

Article Title: In Situ Labeling and Magnetic Resonance Imaging of transplanted Human Hepatic Stem Cells

doi: 10.1007/s11307-010-0422-x

Figure Lengend Snippet: In vivo MR images of unlabeled and contrast labeled hHpSCs in SCID/nod mice. Signal voids indicating the presence of magnetically labeled hHpSC aggregates are absent in the control groups, which include animals that (a) were not transplanted with hHpSCs and did not receive microbeads, (b) were not transplanted with hHpSCs but received microbeads, and (c) were transplanted with hHpSCs but not with microbeads. In contrast, labeled cell aggregates were detected in animals that received hHpSCs and given microbeads at (d) day 6 or (e) day 18 post-transplantation. The enumerated sites correspond to signal voids selected for quantitative analysis and reported in the text.

Article Snippet: In this study, HEA-125 antibodies to EpCAM coupled to magnetic iron microbeads were obtained from Miltenyi Biotech (Bergisch Gladbach, Germany, recently cataloged as CD326 Microbead).

Techniques: In Vivo, Labeling, Control, Transplantation 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: 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