Journal: Cells

Article Title: Generation and Characterization of an Inducible Cx43 Overexpression System in Mouse Embryonic Stem Cells

doi: 10.3390/cells11040694

Figure Lengend Snippet: Generation and characterization of transgenic mouse G4 ES cells with inducible overexpression of connexin (Cx) 43. ( a ) Scheme of the homologous integrated CAG-floxed-Cx43-P2A-mCherry-fragment into the mouse Rs26 locus, enabling the inducible overexpression of Cx43-P2A and the fluorescent protein mCherry. Upon Cre recombinase activity, the loxP flanked stop sites are excised and exogenous Cx43, which is C-terminal-tagged with 21 amino acids of P2A, and mCherry are expressed under control of the CAG-promoter. ( b ) Principle of Cx43 and mCherry co-expression by using the self-cleaving peptide P2A (left side). Transgene expression and Cx43 gap junction formation are depicted schematically in two adjacent cells (right side). ( c ) Scheme of the co-transfection of murine G4 ES cells with the expression vector and zinc finger nucleases and the selection approach of suitable clones. ( d ) PCR analysis of the Rs26 locus revealed correct integration of the transgene. The expected 1325 bp and 4636 bp fragments for the 5′Rs26- and the 3′Rs26 integration, respectively, could be detected. ( e ) Karyotyping of G4 ES cell clones (C) 13, 19, 27, 29, 31, and 52 yielded varying percentages of cells with 40 chromosomes (Chr). ( f ) PCR analysis showed at least one WT allele of the Rs26 locus, indicating a single integration of the transgene. ( g ) Southern blot analysis with α 32 PdCTP labeled probes confirmed the integration of the exogenous Cx43 expression cassette (exogenous Cx43: 5583 bp; endogenous Cx43: 2304 bp, left picture) into the Rs26 locus (integration of the transgene into Rs26: 4171 bp; WT Rs26: 3050 bp, right picture). ( h ) qPCR-based determination of the copy number of the transgene in individual clones: G4 ES cell clones 29 and 31 were found to carry a single copy, whereas clone 13 most likely carries a second copy outside of the Rs26 locus.

Article Snippet: The sequence of the mouse Cx43 cDNA, followed in frame by the DNA sequence of the GSG-P2A peptide and the mCherry cDNA, was synthesized together with flanking FseI sites by GeneArt Gene Synthesis (Thermo Fisher Scientific, Waltham, MA, USA) and subcloned into the pMK-RQ standard vector from Thermo Fisher.

Techniques: Transgenic Assay, Over Expression, Activity Assay, Control, Expressing, Cotransfection, Plasmid Preparation, Zinc-Fingers, Selection, Clone Assay, Southern Blot, Labeling

Journal: Cells

Article Title: Generation and Characterization of an Inducible Cx43 Overexpression System in Mouse Embryonic Stem Cells

doi: 10.3390/cells11040694

Figure Lengend Snippet: Inducible Cx43 overexpression in stably transfected, undifferentiated murine G4 ES cells. ( a ) Stably transfected (CAG-floxSTOP-Cx43-P2A-mCherry-Ai6) G4 ES cells of clones 29 and 31 (Cre − ) were transduced with an AAV2.1-Cre virus (Cre + ) to induce transgene expression. ( b ) Transgenic G4 cells prior to AAV2.1-Cre transduction were mCherry − (left picture, shown for C31), and three days after transduction, most of the colonies were mCherry + (middle picture). Single subclones with strong mCherry expression were isolated, and cells were further cultivated (middle and right pictures, shown for C31). Scale bars 100 µm. ( c ) Immunostainings of Cre + G4 ES cells (subclone 31, upper panel) showed, in contrast to Cre − control cells (C31, lower panel), prominent expression of mCherry and of membrane-associated exogenous Cx43 (P2A + ). Total Cx43 (green), P2A-tagged exogenous Cx43 (white), mCherry (red), and Hoechst (blue). Scale bars 10 µm. ( d ) Western blot analysis of Cre − and Cre + cells of C31 proved inducible Cx43 overexpression; note also mCherry − and P2A expression in the AAV2.1-Cre treated cells. * p value ≤ 0.05.

Article Snippet: The sequence of the mouse Cx43 cDNA, followed in frame by the DNA sequence of the GSG-P2A peptide and the mCherry cDNA, was synthesized together with flanking FseI sites by GeneArt Gene Synthesis (Thermo Fisher Scientific, Waltham, MA, USA) and subcloned into the pMK-RQ standard vector from Thermo Fisher.

Techniques: Over Expression, Stable Transfection, Transfection, Clone Assay, Transduction, Virus, Expressing, Transgenic Assay, Isolation, Control, Membrane, Western Blot

Journal: Cells

Article Title: Generation and Characterization of an Inducible Cx43 Overexpression System in Mouse Embryonic Stem Cells

doi: 10.3390/cells11040694

Figure Lengend Snippet: Inducible Cx43 expression and functional gap junction formation in stably transfected HeLa cells. ( a ) Scheme of single cell dilution of stably transfected (CAG-floxSTOP-Cx43-P2A-mCherry-Ai6, Cre − ) and transduced (AAV2.1-Cre, Cre + ) HeLa cells. Scale bars 100 µm. ( b ) Immunostainings of transgenic HeLa cells confirmed strong expression of exogenous Cx43 and P2A in the plasma membrane of mCherry + cells (Cre + , upper panel), whereas not-transduced control cells were Cx43 − , P2A − , and mCherry − (Cre − , lower panel). Scale bars 10 µm. ( c ) Western blot analysis confirmed expression of Cx43, P2A, and mCherry in Cre + HeLa cells, whereas none of these proteins were detected in Cre − and WT HeLa cells. ( d ) Single Cre + or Cre − HeLa cells were dialyzed via a patch pipette with the small MW dye Alexa350 (349 Da), and dye passage to closely adjacent cells was observed in Cre + cells (upper panel; n = 4). Cre − HeLa control cells did not show passage of Alexa 350 to adjacent cells (lower panel, n = 5). Dialyzed cell marked by a yellow circle; after dye diffusion positive cell colonies marked by a yellow dotted line. Scale bars 100 µm.

Article Snippet: The sequence of the mouse Cx43 cDNA, followed in frame by the DNA sequence of the GSG-P2A peptide and the mCherry cDNA, was synthesized together with flanking FseI sites by GeneArt Gene Synthesis (Thermo Fisher Scientific, Waltham, MA, USA) and subcloned into the pMK-RQ standard vector from Thermo Fisher.

Techniques: Expressing, Functional Assay, Stable Transfection, Transfection, Transgenic Assay, Clinical Proteomics, Membrane, Control, Western Blot, Transferring, Diffusion-based Assay

Journal: Cells

Article Title: Generation and Characterization of an Inducible Cx43 Overexpression System in Mouse Embryonic Stem Cells

doi: 10.3390/cells11040694

Figure Lengend Snippet: Inducible overexpression of functional Cx43 gap junctions in stably transfected 3T3 cells. ( a ) Immunostaining of stably transfected (CAG-floxSTOP-Cx43-P2A-mCherry-Ai6, Cre − ) and transduced (AAV2.1-Cre, Cre + ) mCherry + 3T3-fibroblasts showed strong Cx43 and P2A expression (upper panel). In contrast, Cre − 3T3 cells exhibited much lower Cx43 expression and expressed neither mCherry fluorescence nor P2A. Upper and lower left pictures: Hoechst (blue) and P2A (white); insets: Hoechst (blue), Cx43 (green), mCherry (red), and P2A (white). Scale bars 20 µm. ( b ) Western blot analysis of transgenic Cre + cells proved Cx43 and mCherry (over-)expression but not in control cells. Quantification of Cx43 expression proved its strong overexpression in Cre + 3T3 cells. ( c ) Fluorescence recovery after photobleaching (FRAP) measurements illustrated significantly faster calcein AM dye recovery in transgenic Cre + 3T3-fibroblasts (clone 2) than in either WT or Cre − 3T3-fibroblasts (clone 2); FRAP recovery rates were quantified at 4 and 10 min after bleaching (quantification times are marked by yellow arrows). ( d ) Original fluorescence pictures of 3T3 Cre + , Cre − , and WT fibroblasts, respectively, after calcein AM (0.38 µM) dye loading; bleaching (for 5 s with a 561 nm laser with 2.5 mW intensity) of a single fibroblast (bleached cell marked by a yellow dotted line and a yellow arrow); and dye recovery at 17 s, 37 s, and 57 s after bleaching; calcein AM dye (green). Scale bars 20 µm. ** p value ≤ 0.01, *** p value ≤ 0.001, **** p value ≤ 0.0001, ns (not significant) p value > 0.05.

Article Snippet: The sequence of the mouse Cx43 cDNA, followed in frame by the DNA sequence of the GSG-P2A peptide and the mCherry cDNA, was synthesized together with flanking FseI sites by GeneArt Gene Synthesis (Thermo Fisher Scientific, Waltham, MA, USA) and subcloned into the pMK-RQ standard vector from Thermo Fisher.

Techniques: Over Expression, Functional Assay, Stable Transfection, Transfection, Immunostaining, Expressing, Fluorescence, Western Blot, Transgenic Assay, Control

Journal: Cells

Article Title: Generation and Characterization of an Inducible Cx43 Overexpression System in Mouse Embryonic Stem Cells

doi: 10.3390/cells11040694

Figure Lengend Snippet: Inducible Cx43 (over-)expression in stably transfected murine G4 ES cells (clone 31), in vitro differentiation into cardiomyocytes. ( a ) In vitro differentiation scheme of murine ES cells into spontaneously beating EBs (adapted from Boheler et al. ). ( b ) The mCherry + EBs derived from the transgenic Cre + G4 ES cells of clone 31 (upper picture) and transgenic mCherry − control EBs (lower picture). Scale bars 100 µm. ( c ) Western blot analysis of EBs from Cre + and Cre − G4 ES cells confirmed inducible Cx43 overexpression, as underscored by P2A-tagged Cx43 and mCherry expression. ( d ) Immunostainings of EBs illustrated clusters of cardiac α-actinin + (white) cardiomyocytes in both Cre + (upper left picture) and Cre − control EBs (lower left picture). Immunostainings yielded strong Cx43 (green, upper left picture), P2A (white), and mCherry (red) (over-)expression (upper middle picture) in Cre + EBs. Similar apoptotic areas preferentially in the center of Cre + and Cre − control EBs (right pictures) were found. Scale bars 10 µm.

Article Snippet: The sequence of the mouse Cx43 cDNA, followed in frame by the DNA sequence of the GSG-P2A peptide and the mCherry cDNA, was synthesized together with flanking FseI sites by GeneArt Gene Synthesis (Thermo Fisher Scientific, Waltham, MA, USA) and subcloned into the pMK-RQ standard vector from Thermo Fisher.

Techniques: Over Expression, Stable Transfection, Transfection, In Vitro, Derivative Assay, Transgenic Assay, Control, Western Blot, Expressing

Journal: Cells

Article Title: Generation and Characterization of an Inducible Cx43 Overexpression System in Mouse Embryonic Stem Cells

doi: 10.3390/cells11040694

Figure Lengend Snippet: Functional impact of Cx43 overexpression in murine G4 ES cell-derived EBs; targeting of murine neonatal cardiomyocytes (NNCMs) with an AAV2.6-Cx43 overexpression virus. ( a ) Fluorescent and brightfield pictures of Cre + and Cre − EB-derived cell clusters at one day after dissociation and plating. Scale bars 100 µm. Video-based monitoring of spontaneous beating rates showed that Cre + cell clusters (red) exhibited significantly higher and more stable beating rates compared to Cre − controls (black). **** p value ≤ 0.0001. ( b ) Scheme depicting the isolation and enrichment of NNCMs. ( c ) NNCMs were cultivated and transduced with the AAV2.6-CAG-Cx43-P2A-mCherry (AAV2.6-Cx43) virus; note strong mCherry fluorescence (picture). Transduction efficiency of AAV2.6-Cx43 virus in NCCMs (graph). ( d ) Immunostainings of transduced NNCMs showed Cx43 (green), P2A (white), and mCherry (red) expression (upper panel), whereas non-transduced NNCMs displayed less Cx43 and neither P2A nor mCherry (second panel) expression. The mCherry + (red) and cardiac α-actinin + (white cross-striation) immunostainings (third panel) proved successful targeting of NNCMs with the AAV2.6-Cx43 virus; non-transduced NNCMs showed α-actinin, but less Cx43 staining and no mCherry signal. NNCMs lack intercalated discs; note the diffuse expression pattern of Cx43 in the cell membrane. Scale bars 10 µm.

Article Snippet: The sequence of the mouse Cx43 cDNA, followed in frame by the DNA sequence of the GSG-P2A peptide and the mCherry cDNA, was synthesized together with flanking FseI sites by GeneArt Gene Synthesis (Thermo Fisher Scientific, Waltham, MA, USA) and subcloned into the pMK-RQ standard vector from Thermo Fisher.

Techniques: Functional Assay, Over Expression, Derivative Assay, Virus, Isolation, Transduction, Fluorescence, Expressing, Staining, Membrane

Journal: Journal of Neurophysiology

Article Title: Isoform-specific phosphorylation-dependent regulation of connexin hemichannels

doi: 10.1152/jn.00575.2015

Figure Lengend Snippet: Effect of protein kinase C (PKC) inhibition on hemichannel permeability to ethidium. A: ethidium (Eth) uptake [in arbitrary units (a.u.)] was determined in uninjected oocytes and oocytes expressing connexin (Cx)30 or Cx43 exposed to control solution or divalent cation-free solution (DCFS) for 60 min in the presence or absence of the PKC inhibitors bisindolylmaleimide (BIM; 5 μM) and chelerythrine (CHEL; 10 μM) [n = 5 for Cx43- and Cx30-expressing oocytes and n = 6 for uninjected (Uninj) oocytes]. B: as in A but without ethidium present (n = 3 for Cx43- and Cx30-expressing oocytes and n = 4 for uninjected oocytes). For comparison, dashed line indicates fluorescence count obtained in the presence of ethidium in uninjected oocytes in the control solution from A. Statistical significance was tested with mixed-model analysis of variance, with Bonferroni (A) or Dunnett's (B) post hoc tests. *Statistical significance vs. corresponding group in control solution; #statistical significance vs. vehicle-treated oocytes. Note that the statistics were carried out on pooled data for control solutions in A or for all groups in B; please see explanation in results. For clarity the symbols in B are shown adjacent to the key. *P < 0.05, ##P < 0.01, ***/###P < 0.001. ns, Not significant.

Article Snippet: In vitro transcription. cDNA encoding mouse Cx30 (mCx30; obtained from Klaus Willecke, Bonn University) and rat Cx43 (rCx43; obtained from Zealand Pharma) were subcloned into the expression vector pXOOM and their sequence verified.

Techniques: Inhibition, Permeability, Expressing, Fluorescence

Journal: Journal of Neurophysiology

Article Title: Isoform-specific phosphorylation-dependent regulation of connexin hemichannels

doi: 10.1152/jn.00575.2015

Figure Lengend Snippet: Regulation of Cx30 but not Cx43 hemichannels by PKC activation. Ethidium uptake was determined after exposing Cx30-expressing oocytes (A), Cx43-expressing oocytes (B), or uninjected oocytes (B, inset) to DCFS for 10, 20, 40, and 60 min with vehicle, phorbol 12-myristate 13-acetate (PMA), or Gd3+ present in the solution (n = 5). C: summary of the ethidium uptake after 60 min. Statistical significance was tested with repeated-measures 2-way ANOVA with Bonferroni post hoc test. ***P < 0.001.

Article Snippet: In vitro transcription. cDNA encoding mouse Cx30 (mCx30; obtained from Klaus Willecke, Bonn University) and rat Cx43 (rCx43; obtained from Zealand Pharma) were subcloned into the expression vector pXOOM and their sequence verified.

Techniques: Activation Assay, Expressing

Journal: Journal of Neurophysiology

Article Title: Isoform-specific phosphorylation-dependent regulation of connexin hemichannels

doi: 10.1152/jn.00575.2015

Figure Lengend Snippet: Membrane expression of Cx30 and Cx43 after PMA stimulation. A: uninjected oocytes and oocytes expressing Cx30 or Cx43 were exposed to DCFS with PMA or vehicle for 1 h prior to plasma membrane purification. The purified plasma membrane fraction was subjected to SDS gel electrophoresis and subsequent immunoblotting with anti-Cx30 or anti-Cx43 antibodies. Representative Western blots are shown for Cx30 (left) and Cx43 (right). Summarized densitometry illustrates the fractional connexin surface expression after PMA treatment relative to that obtained in control solution (n = 5). B: Cx30-expressing oocytes were treated as above prior to total membrane purification and Western blotting. Representative blot (top) and summarized densitometry (bottom) illustrate the Cx30 abundance after PMA treatment relative to that obtained in control solution (n = 5). Statistical significance was tested with 1-sample t-test against the hypothetical value of 1.

Article Snippet: In vitro transcription. cDNA encoding mouse Cx30 (mCx30; obtained from Klaus Willecke, Bonn University) and rat Cx43 (rCx43; obtained from Zealand Pharma) were subcloned into the expression vector pXOOM and their sequence verified.

Techniques: Expressing, Purification, SDS-Gel, Electrophoresis, Western Blot

Journal: Journal of Neurophysiology

Article Title: Isoform-specific phosphorylation-dependent regulation of connexin hemichannels

doi: 10.1152/jn.00575.2015

Figure Lengend Snippet: Mimic of (de)phosphorylation of Ser368 in the Cx43 COOH terminus does not affect hemichannel activity. The Cx43-S368 residue was mutated to alanine (Cx43-S368A) or aspartate (Cx43-S368D). Ethidium uptake was quantified after 1 h in control solution or DCFS. Statistical significance was tested with 2-way ANOVA, repeated measurements with Bonferroni post hoc test. *P < 0.05.

Article Snippet: In vitro transcription. cDNA encoding mouse Cx30 (mCx30; obtained from Klaus Willecke, Bonn University) and rat Cx43 (rCx43; obtained from Zealand Pharma) were subcloned into the expression vector pXOOM and their sequence verified.

Techniques: De-Phosphorylation Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Induction of Apoptosis by PQ1, a Gap Junction Enhancer that Upregulates Connexin 43 and Activates the MAPK Signaling Pathway in Mammary Carcinoma Cells

doi: 10.3390/ijms17020178

Figure Lengend Snippet: Modulation of connexin 43 expression affects proliferation and viability of FMC2u cells. ( A ) Graphical representation of fold protein induction from western blot analysis of Cx43 in FMC2u cells transfected with GFP-Cx43 cDNA vectors; ( B ) Graphical representation of fold protein induction from western blot analysis of Cx43 in FMC2u cells transfected with siRNA of Cx43; ( C ) Viability and ( D ) Total cell count either overexpressing (OE) Cx43 or with silenced (siRNA) Cx43 compared to normal unmodified cells; ( E ) Graphical representation of cleaved caspase-3 expression in FMC2u cells post transfection. All experiments conducted with a sample size of n = 3. * p -value < 0.05 compared to respective transfection control.

Article Snippet: cDNA from human Cx43 was purchased from Addgene (Cambridge, MA, USA; plasmid #40907).

Techniques: Expressing, Western Blot, Transfection, Cell Counting, Control

Journal: International Journal of Molecular Sciences

Article Title: Induction of Apoptosis by PQ1, a Gap Junction Enhancer that Upregulates Connexin 43 and Activates the MAPK Signaling Pathway in Mammary Carcinoma Cells

doi: 10.3390/ijms17020178

Figure Lengend Snippet: Modulation of connexin 43 expression affects GJIC. ( A ) Gap junction activity of FMC2u determined by scrape load dye transfer after transfection with empty vector (control), GFP-Cx43 cDNA vector, or siRNA Cx43. A graphical representation of dye transfer in µm was shown next to the images. Red lines indicate a cross sectional cut. Lucifer yellow was used as a gap junctional dye and Rhodamine-dextran used to mark the cut site. Green fluorescence indicates the passage of dye form the cutting site. Images taken at 4×, 10×, and 20×. Scale bar = 100, 100, 50 µm, respectively. Graphical representation of ZO-1 expression in Cx43-immunoprecipitated FMC2u cells at 0, 6, 12, 24, 36, and 48 h post transfection; ( B ) Induced overexpression of Cx43 and ( C ) a silencing of Cx43. All experiments conducted with a sample size of n = 3. * p -value < 0.05 compared to respective transfection control.

Article Snippet: cDNA from human Cx43 was purchased from Addgene (Cambridge, MA, USA; plasmid #40907).

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Control, Fluorescence, Immunoprecipitation, Over Expression

Journal: International Journal of Molecular Sciences

Article Title: Induction of Apoptosis by PQ1, a Gap Junction Enhancer that Upregulates Connexin 43 and Activates the MAPK Signaling Pathway in Mammary Carcinoma Cells

doi: 10.3390/ijms17020178

Figure Lengend Snippet: Altering connexin 43 affects expression of signaling molecules involved in cellular survival. ( A ) Raw and ( B – E ) graphical representation of western blot analysis of cRaf, phosphorylated (P) and unphosphorylated MAPK (p44/42 and p38), and Bcl-2 expression levels relative to loading control in FMC2u cells transfected with either cDNA of Cx43 to induce overexpression ( Left ) or siRNA to silence Cx43 ( right ). Actin used as a loading control. n = 3. * p -value < 0.05 compared to respective transfection control.

Article Snippet: cDNA from human Cx43 was purchased from Addgene (Cambridge, MA, USA; plasmid #40907).

Techniques: Expressing, Western Blot, Control, Transfection, Over Expression