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: Korean Circulation Journal

Article Title: Preventive Effects of the Angiotensin-II Receptor Blocker on Atrial Remodeling in an Ischemic Heart Failure Model of Rats

doi: 10.4070/kcj.2013.43.10.686

Figure Lengend Snippet: Even distribution of connexin 43 protein on the intercalated disc was observed in the sham group (A: magnification: ×400). Connexin 43 protein stain was not observed on the intercalated disc in the heart failure group (B: magnification: ×400). A slight connexin 43 protein stain was observed on the intercalated disc in the heart failure-ARB group (C: magnification: ×400). Western blot of connexin 43 protein showed a lower expression in the heart failure-ARB group (D). ARB: angiotensin-II receptor blocker.

Article Snippet: The immunohistochemical stain for connexin 43 (rabbit polyclonal anti-Cx43 antibody, 1/100, Zymed-laboratories, CliniSciences, France) showed a significant amount of plasmalemmal distribution of connexin 43 in the heart failure group.

Techniques: Staining, Western Blot, Expressing

Journal: Oncology reports

Article Title: Simvastatin protects Sertoli cells against cisplatin cytotoxicity through enhanced gap junction intercellular communication.

doi: 10.3892/or.2015.4192

Figure Lengend Snippet: Figure 2. Inhibition of junctional communication by Cx43 siRNA and pharmacological inhibitor TPA. (A) Western blotting probed with anti-Cx43 antibody shows the decrease of the Cx43 level in siRNA-transfected cells. (B and C) Cx43-siRNA and TPA reduce dye coupling through gap junction as measured by parachute dye-coupling assay. Columns, mean for five experiments; bars, SEM. **, Significantly different from control, P<0.01.

Article Snippet: Individual membranes were probed with monoclonal antiCx43 antibody produced in mouse (1:4,000; Sigma-Aldrich) and monoclonal antibodies against p-Cx43 (ser368) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Inhibition, Western Blot, Transfection, Control

Journal: Oncology reports

Article Title: Simvastatin protects Sertoli cells against cisplatin cytotoxicity through enhanced gap junction intercellular communication.

doi: 10.3892/or.2015.4192

Figure Lengend Snippet: Figure 3. Simvastatin attenuates cisplatin toxicity at high cell density and this effect was blocked by Cx43-siRNA. (A) Clonogenic survival of cells pretreated with 10 µM simvastatin for 3 h, and co-incubated with 5 µM cisplatin for 1 h at high and low cell density. (B) Clonogenic survival of cells pretreated with a range of concentrations of simvastatin for the indicated periods, followed by co-incubation with 5 µM cisplatin for 1 h at high cell density. (C) Clonogenic survival of siRNA-transfected cells pretreated with 10 µM simvastatin for 3 h, and co-incubated with 5 µM cisplatin for 1 h at high and low cell density. Columns, mean for five experiments; bars, SEM. *, Significantly different from control, P<0.05; #, significantly different from the cisplatin bar, P<0.05.

Article Snippet: Individual membranes were probed with monoclonal antiCx43 antibody produced in mouse (1:4,000; Sigma-Aldrich) and monoclonal antibodies against p-Cx43 (ser368) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Incubation, Transfection, Control

Journal: Oncology reports

Article Title: Simvastatin protects Sertoli cells against cisplatin cytotoxicity through enhanced gap junction intercellular communication.

doi: 10.3892/or.2015.4192

Figure Lengend Snippet: Figure 4. Effects of simvastatin on gap junction intercellular communication, Cx43 immunostaining. (A) The dye spread of cells exposed to increasing concentrations of simvastatin for 4 h as assayed by parachute dye-coupling assay. (B) Immunostaining for Cx43 in cells with or without 10 µM simvastatin treatment. Magnification, x400.

Article Snippet: Individual membranes were probed with monoclonal antiCx43 antibody produced in mouse (1:4,000; Sigma-Aldrich) and monoclonal antibodies against p-Cx43 (ser368) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Immunostaining

Journal: Oncology reports

Article Title: Simvastatin protects Sertoli cells against cisplatin cytotoxicity through enhanced gap junction intercellular communication.

doi: 10.3892/or.2015.4192

Figure Lengend Snippet: Figure 5. Effects of simvastatin on Cx43 expression and p-Cx43 (ser368) expression. (A) Western blot analysis of Cx43 expression in cells treated with varying concentrations of simvastatin. (B) Western blotting showing p-Cx43 (ser368) levels in cells following treatment with 10 µM simvastatin for varying time periods. Bar graphs are derived from the densitometric scanning of the blots and shows a comparison of the level of Cx to β-tubulin density ratio. Columns, mean from four experiments; bars, SEM. *, Significantly different from control.

Article Snippet: Individual membranes were probed with monoclonal antiCx43 antibody produced in mouse (1:4,000; Sigma-Aldrich) and monoclonal antibodies against p-Cx43 (ser368) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing, Western Blot, Derivative Assay, Comparison, Control

Journal: Nature Communications

Article Title: Physiological synaptic activity and recognition memory require astroglial glutamine

doi: 10.1038/s41467-022-28331-7

Figure Lengend Snippet: a Representative confocal images showing close proximity of Cx43 (cyan) in GFAP-eGFP positive astrocytes (yellow) to presynaptic structures immunolabeled for VGlut1 (magenta). Higher magnification images of a region containing astroglial processes (blue square) are shown in the middle row. Arrowheads denote points of close contact. Masks showing co-localized area of GFAP (yellow) and Cx43 (cyan) as total Cx43 (binary inverse image) and co-localized area of total Cx43 (cyan) and VGlut1(magenta) as presynaptic Cx43 (binary inverse image). b Bar graph (mean ± SEM) showing % Perisynaptic Cx43 normalized to total Cx43 area ( n = 13 fields, 3 independent experiments). c Schematic illustration of co-purification of perisynaptic astroglial processes in crude synaptosomes. d Representative western blots showing an enrichment of Cx43 protein in synaptosomal preparations (Syn) compared to total hippocampal lysates (Hip) in wild type (+/+), but not in glial conditional Cx43 knockout (−/−) mice. GAPDH was used as a loading control. e Representative high magnification electron micrographs showing the presence of Cx43 protein labeled by immunogold particles in astroglial processes near synaptic complexes. f Distribution histogram of distance between Cx43 gold grains and the nearest active zone. Scale bars: a 5 µm, e 0.5 µm (left), 0.3 µm (right). Representative images ( a ) are from replicates described in ( b ); d are from n = 3 replicates; and e are from n = 8 replicates. Source data are provided as a Source data file.

Article Snippet: The following primary antibodies were used: polyclonal chicken anti-GFP (1:500, AB13970, Abcam), monoclonal mouse anti-VGlut1 (1:200, 135511, Synaptic Systems), monoclonal mouse anti-Cx43 (1:500, 610-062, BD Biosciences), polyclonal rabbit anti-Cx43 (1:500, 71-2200, Zymed Laboratories), monoclonal mouse anti-GFAP (1:500, G3893, Sigma), and monoclonal mouse anti-GAPDH-peroxidase (1:1000, G9295, Sigma).

Techniques: Immunolabeling, Copurification, Western Blot, Knock-Out, Labeling

Journal: Nature Communications

Article Title: Physiological synaptic activity and recognition memory require astroglial glutamine

doi: 10.1038/s41467-022-28331-7

Figure Lengend Snippet: Acute hippocampal slices were loaded with ethidium bromide (EtBr) under different experimental conditions. a Sample images of EtBr uptake (magenta) in hippocampal GFAP-immunolabeled astrocytes (green) are shown for Control, Stim (10 Hz, 30 s every 3 min for 20 min) in the absence or presence of the Cx43 HC blocker Gap26 or a Gap26 scramble version (Src). Higher magnifications of the CA1 stratum radiatum subregion are shown in bottom two rows. b Schematic illustrating stimulation of hippocampal Schaffer collaterals and EtBr uptake in neighboring astrocytes. c Quantification of EtBr uptake normalized to 100% control (dotted line) is shown. Stimulation-enhanced EtBr uptake by nearly 2-fold (mean ± SEM; Control, n = 6; Stim, n = 7, p = 0.0002 between Control and Stim, one-sampled t -test). This enhanced uptake was not observed in the presence of Gap26 (Stim + Gap26, n = 4, p < 0.0001 between Stim and Stim+Gap26, one-way ANOVA with Bonferroni’s post hoc test; p = 0.0168 with control, one-sampled t -test) but persisted with Src (Stim + Src, n = 4, p = 0.6857 between Stim and Stim+Src, one-way ANOVA with Bonferroni’s post hoc test; p = 0.0125 with control, one-sampled t -test), while Gap26 alone decreases EtBr uptake from control level ( n = 5, p = 0.014, one-sampled t -test) but not Src ( n = 5, p = 0.4443, one-sampled t -test). Scale bars: a 450 µm (top), 50 µm (middle and bottom). Asterisks indicate statistical significance (*** p < 0.001; * p < 0.05). Source data are provided as a Source data file.

Article Snippet: The following primary antibodies were used: polyclonal chicken anti-GFP (1:500, AB13970, Abcam), monoclonal mouse anti-VGlut1 (1:200, 135511, Synaptic Systems), monoclonal mouse anti-Cx43 (1:500, 610-062, BD Biosciences), polyclonal rabbit anti-Cx43 (1:500, 71-2200, Zymed Laboratories), monoclonal mouse anti-GFAP (1:500, G3893, Sigma), and monoclonal mouse anti-GAPDH-peroxidase (1:1000, G9295, Sigma).

Techniques: Immunolabeling

Journal: Nature Communications

Article Title: Physiological synaptic activity and recognition memory require astroglial glutamine

doi: 10.1038/s41467-022-28331-7

Figure Lengend Snippet: Acute hippocampal slices were loaded with ethidium bromide (EtBr) under stimulated conditions in the absence or presence of various blockers. a , b Sample images of EtBr uptake (magenta) in hippocampal GFAP-immunolabeled astrocytes (green) are shown in ( a ) and quantified in ( b ). Stimulation-enhanced uptake was blocked in the presence of NBQX + CPP (20 μM, n = 6, p = 0.0112 with wild-type control, +/+, n = 11, one-way ANOVA with Bonferroni’s post hoc test), but not LY341495 (20 μM; n = 5, p = 0.2433 with +/+, one-way ANOVA with Bonferroni’s post hoc test), indicating the specific involvement of ionotropic glutamate receptor activity. This stimulation-dependent uptake was also blocked in the presence of BaCl 2 (200 μM; n = 7, p = 0.0004 with +/+, one-way ANOVA with Bonferroni’s post hoc test) and in acute slices prepared from Kir4.1−/− mice ( n = 5, p = 0.0086 with +/+, one-way ANOVA with Bonferroni’s post hoc test), indicating the specific involvement of Kir4.1 activity. c , d Incubation of acute slices with either 2 mM K + or 1 μM glutamate (Glut) alone enhanced basal EtBr uptake (K + + Src, n = 9, 3 experiments, p = 0.0061; Glut+Src, n = 9, 3 experiments, p = 0.0056 with Src, n = 14, 5 experiments; Kruskal–Wallis test with Dunn’s post hoc test) in a Cx43 HC-dependent manner (K + + Gap26, n = 20, 7 experiments, p < 0.0001 with K + + Src; Glut + Gap26, n = 11, 5 experiments, p < 0.0001 with Glut+Src; Kruskal–Wallis test with Dunn’s post hoc test). In Kir4.1−/− hippocampal slices, neither K + nor Glut were able to enhance EtBr uptake (K + Kir4.1−/−, n = 49, 17 experiments, p < 0.0001 with K + +/+, n = 22, 8 experiments; Glut Kir4.1−/−, n = 27, 9 experiments, p = 0.0001 with Glut +/+, n = 23, 8 ex p eriments, Mann–Whitney test). Mean ± SEM in ( b ) and ( d ). Scale bars: a and c , 50 µm. Asterisks indicate statistical significance (*** p < 0.001; ** p < 0.01; * p < 0.05). NBQX = 2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo[f]chinoxalin-2,3-dion; CPP = (3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid. Source data are provided as a Source data file.

Article Snippet: The following primary antibodies were used: polyclonal chicken anti-GFP (1:500, AB13970, Abcam), monoclonal mouse anti-VGlut1 (1:200, 135511, Synaptic Systems), monoclonal mouse anti-Cx43 (1:500, 610-062, BD Biosciences), polyclonal rabbit anti-Cx43 (1:500, 71-2200, Zymed Laboratories), monoclonal mouse anti-GFAP (1:500, G3893, Sigma), and monoclonal mouse anti-GAPDH-peroxidase (1:1000, G9295, Sigma).

Techniques: Immunolabeling, Activity Assay, Incubation, MANN-WHITNEY

Journal: Nature Communications

Article Title: Physiological synaptic activity and recognition memory require astroglial glutamine

doi: 10.1038/s41467-022-28331-7

Figure Lengend Snippet: a Representative confocal (dark background) and thresholded binary (white background) images of hippocampal CA1 astrocytes dialyzed with RhGln (0.8 mM, 20 min) via the patch pipette under control or stimulated (10 Hz, 30 s every 3 min for 20 min) conditions in acute slices obtained from: wild-type mice (+/+), glial conditional Cx43 knockout mice (−/−) or wild-type mice exposed to Gap26 (+/+ Gap26) or Gap26 scramble (+/+Src) peptides. The binary images were quantified by Sholl analysis ( b ) and total punctate area ( c ). In +/+ mice, repetitive synaptic stimulation strongly increased the punctate RhGln-labeling compared to control as shown by both Sholl analysis (+/+: Ct, n = 12; Stim, n = 9, p < 0.0001, two-way ANOVA in b ) and total punctate area ( p < 0.0001 between Ct and Stim in +/+, one-way ANOVA with Bonferroni’s post hoc test in c ). This was abolished in −/− mice (−/−: Ct, n = 7; Stim, n = 5, p = 0.0826, two-way ANOVA in b ; p > 0.999 between Ct and Stim in −/−, and p = 0.0002 between +/+ Stim and −/− Stim, one-way ANOVA with Bonferroni’s post hoc test in c ) or in the presence of Gap26 (+/+ Gap26: Ct, n = 8; Stim, n = 5, p = 0.0651, two-way ANOVA in b ; p > 0.999 between Ct and Stim in +/+ Gap26, and p < 0.0001 between Stim of +/+ and +/+ Gap26, one-way ANOVA with Bonferroni’s post hoc test in c ), but unchanged in the presence of the scramble Gap26 peptide (+/+Src: Ct, n = 5; Stim, n = 4, p < 0.0001, two-way ANOVA in b ; p = 0.025 between Ct and Stim with +/+ Src, and p < 0.0001 between Stim of +/+ Gap26 and +/+ Src, one-way ANOVA with Bonferroni’s post hoc test in c ). Gap26 alone also inhibited the spread of glutamine, suggesting a basal transfer of glutamine into synaptic structures which is dependent on Cx43 HC activity ( p < 0.0001 between +/+ Gap26 Ct and +/+ Gap26 Stim, two-way ANOVA in b ). Mean ± SEM in ( b ), ( c ). Scale bar: a 20 µm. Asterisks indicate statistical significance (*** p < 0.001; * p < 0.05). Source data are provided as a Source data file.

Article Snippet: The following primary antibodies were used: polyclonal chicken anti-GFP (1:500, AB13970, Abcam), monoclonal mouse anti-VGlut1 (1:200, 135511, Synaptic Systems), monoclonal mouse anti-Cx43 (1:500, 610-062, BD Biosciences), polyclonal rabbit anti-Cx43 (1:500, 71-2200, Zymed Laboratories), monoclonal mouse anti-GFAP (1:500, G3893, Sigma), and monoclonal mouse anti-GAPDH-peroxidase (1:1000, G9295, Sigma).

Techniques: Transferring, Knock-Out, Labeling, Activity Assay

Journal: Nature Communications

Article Title: Physiological synaptic activity and recognition memory require astroglial glutamine

doi: 10.1038/s41467-022-28331-7

Figure Lengend Snippet: a Sample image of the hippocampus of Cx43−/− mice injected intra-hippocampally with rAAV2/9-GFAP-Cx43-GFP virus, showing numerous cells expressing Cx43-GFP in the CA1 area. The blue box is magnified on the right. b RhGln (magenta) was loaded into a Cx43-GFP expressing astrocyte (yellow) via a patch pipette as shown. Arrow head indicates the patched cell. c Sample images after immunostaining showing specific expression of Cx43 (cyan) in Cx43-GFP-positive (yellow) astrocytes (GFAP, magenta). The yellow box is magnified in the bottom row. Solid and dotted white lines outline GFP-positive and -negative astrocytes, respectively. d – g Cx43−/− mice first received either rAAV2/9-GFAP-Cx43-GFP (−/− Cx43 Rescue, d left, e and g ) or rAAV2/9-GFAP-GFP (−/− GFP Control, d right, f and g ) virus. Hippocampal astrocytes were then dialyzed with RhGln under either control or synaptic stimulation (10 Hz, 30 s) conditions for 20 min. Both representative confocal (dark background) and thresholded binary (white background) images are shown in ( d ) for each condition. The binary images were quantified by Sholl analysis ( e , f ) and total punctate area ( g ). The stimulation-induced transfer of RhGln was rescued in Cx43−/− mice ( n = 5) by restoring Cx43 expression selectively in astrocytes via viral infection shown by both Sholl analysis (−/− Cx43 rescue, n = 4, p < 0.0001, two-way ANOVA for e ) and total punctate area ( p = 0.0031 between Ct and Stim with −/− Cx43 Rescue, one-way ANOVA with Bonferroni’s post hoc test for g ) as compared to the GFP control infection (−/− GFP Ct, n = 3; Stim, n = 3, p = 0.9856, two-way ANOVA for f , p > 0.999 between Ct and Stim with −/−GFP Control and p < 0.0001 between Stim of −/− Cx43 Rescue and −/− GFP Control, one-way ANOVA with Bonferroni’s post hoc test for g ). Mean ± SEM in ( e )–( g ). Scale bars: a 200 µm (left) and 50 µm (right); b 50 µm (top) and 20 µm (bottom); c , d 20 µm. Asterisks indicate statistical significance (*** p < 0.001; ** p < 0.01). Representative images a , b from replicates described in ( g ); c are from n = 3 replicates. Source data are provided as a Source data file.

Article Snippet: The following primary antibodies were used: polyclonal chicken anti-GFP (1:500, AB13970, Abcam), monoclonal mouse anti-VGlut1 (1:200, 135511, Synaptic Systems), monoclonal mouse anti-Cx43 (1:500, 610-062, BD Biosciences), polyclonal rabbit anti-Cx43 (1:500, 71-2200, Zymed Laboratories), monoclonal mouse anti-GFAP (1:500, G3893, Sigma), and monoclonal mouse anti-GAPDH-peroxidase (1:1000, G9295, Sigma).

Techniques: Injection, Expressing, Transferring, Immunostaining, Infection

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: A) Schematics of full length Cx43 and αCT1 peptide. B) αCT1 interaction with ZO-1 PDZ domains as indicated by EDC zero-length cross-linking to GST fusion PDZ1, PDZ2 and PDZ3 polypeptides and neutravidin labeling of biotin-tagged peptide at concentrations of 5, 25 and 50 μM. The deletion of the CT Isoleucine (I) in αCT1-I renders this peptide incompetent to interact with the ZO-1 PDZ2 domain. C) Coomassie blue gel of EDC cross-linked products of kinase reaction mixtures containing GST-Cx43 CT and PKC-ε, with (αCT1) and without (Vehicle) αCT1. The fainter band above GST-Cx43 bands (indicated by lines) in the αCT1 lanes were cut from gels and analyzed by Tandem Mass Spectrometry (MS/MS). The boxes to right of gel show Cx43 CT peptides identified by MS/MS as being cross-linked to αCT1. D) Tandem mass spectrum of a quintuply charged crosslinked peptide (m/z: 674.1) between Cx43 345-366 (a-chain) and αCT1 peptide through Cx43 K346 and E8 in αCT1 (b-chain). Only the b-and y-sequence specific ions are labeled. Arrow indicates ion (b a5 2+ ) consistent with cross-linkage between Cx43 CT lysine K346 and the glutamic acid (E) residue of αCT1 at position −1.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Labeling, Mass Spectrometry, Tandem Mass Spectroscopy, Sequencing

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: Blots of EDC cross-linked products of kinase reaction mixtures containing GST-Cx43 CT, GST-Cx43 CT QQ/KK in which the lysine (K) residues were mutated to neutral glutamines (Q), PKC-ε and αCT1 (at 5, 10 and 25 μM) and a scrambled αCT1 (M4 scr) variant at the same concentrations. Only αCT1 is seen to be covalently linked by EDC to Cx43 CT in a concentration-dependent manner.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Variant Assay, Concentration Assay

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: A) Schematics of Cx43 and the secondary structure of Cx43 CT from amino acid residues Glycine252 (G252) through to Isoleucine 382 (I382). The depiction of secondary structure in 2A has been modified from a diagram originally provided by Sosinsky and co-workers . B) ZDOCK and C) Schrodinger molecular modeling software analysis of the structure of a proposed αCT1-Cx43 CT complex. The protonated structure of αCT1 peptide and Cx43 CT (PDB:1r5s), constrained by a salt-bridge interaction between K346 in the Cx43 CT and the glutamic acid (E) at position −1 of αCT1. The αCT1-Cx43 interaction shown represents that based on the lowest energy minimization score determined in the model. D) Schrodinger molecular modeling software, a 2D map of αCT1-Cx43 CT in anti-parallel orientation showing location of amino acids predicted to bond to each other and the type of bond that is predicted to occur.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Modification, Software

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: SPR was used to analyze interactions of biotin-αCT1 and biotin-αCT1 variant peptides, immobilized to streptavidin-coated chips, with the Cx43 CT (Cx43-CT: amino acids 255 to 382) and Cx43 CT-KK/QQ as analytes, respectively. The mean of three runs is plotted for each analyte concentration. The exposure of the sensor chip to the specific analyte is indicated by the gray area. Sensorgrams obtained for: A) Cx43 CT and biotin-αCT1. B) Cx43 CT-KK/QQ and biotin-αCT1. C) Cx43 CT and biotin-M1 AALAI. D) Cx43 CT-KK/QQ and biotin-M1 AALAI. E) Cx43 CT and biotin-M3 DDLAI. F) Cx43 CT-KK/QQ and biotin-M3 DDLAI.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Variant Assay, Concentration Assay

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: A) Melt curves (top) and first derivative of melt curves (bottom) for ZO-1 PDZ2 at 500 μg/mL in combination αCT1 at concentrations of 25, 50 and 100 μM. B) Temperature maxima (Tm) from Boltzman curves from left-to-right of Cx43 CT (Cx43-CT: amino acids 255 to 382) alone, Cx43 CT in combination with αCT1, and the αCT1 variants including: M1 AALAI, M2 AALEI, M3 DDLAI, M4 scrambled, αCT-I and αCT11. αCT1, αCT1-I and αCT11 show similar abilities to destabilize (i.e., significantly decrease the Tm of) Cx43 CT. **p<0.01, *** p<0.002, N=6. C) Temperature maxima (Tm) from Boltzman curves from left-to-right of PDZ2 alone, and PDZ2 in combination with αCT1 and αCT1variants including αCT1 variants including: M1 AALAI, M2 AALEI, M3 DDLAI, M4 scrambled, αCT-I and αCT11. M3 DDLAI, αCT1, and αCT11 show similar abilities to stabilize (i.e., significantly increase the Tm of) PDZ2. **p<0.01, ***p<0.002, N=6

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques:

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: A) Blots of Cx43-pS368 (top) and total Cx43 (bottom) in kinase reactions mixtures including no-kinase controls with substrate (Cx43-CT: amino acids 255 to 382), but no PKC-ε (PKC-minus); Cx43-CT substrate with PKC-ε (PKC-plus); and mixtures containing PKC-ε, Cx43 CT, and biotin-tagged αCT1, biotin-tagged αCT1 mutant peptides with alanine substitutions (M1 AALAI, M2 AALEI, M3 DDLAI) and biotin-tagged M4 scrambled. Peptides are at 20 μM. B) Blots of Cx43-pS368 (top) and total Cx43 (bottom) in kinase reactions mixtures including no-kinase controls with Cx43 CT substrate, but no PKC-ε (PKC-minus); Cx43-CT substrate with PKC-ε (PKC-plus); and mixtures containing PKC-ε, Cx43 CT, and biotin-αCT1, biotin-αCT1-I or biotin-αCT11 (RPRPDDLEI with no antennapedia sequence at peptide NT) and biotin-M4 scrambled peptide. Peptides are at 20 μM. C) Chart showing that the ability of unmodified αCT1 and the Cx43 CT interaction-competent peptides biotin-αCT1-I or biotin-αCT11 to induce S368 phosphorylation was 3-5 fold greater than that of non-Cx43 CT interacting peptides. * p<0.05, ** p<0.01, *** p<0.002, N=5 αCT1 and M4, other peptides N=3.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Mutagenesis, Sequencing

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: Langendorff ischemia-reperfusion (I/R) injury protocols were performed on adult mouse hearts instrumented to monitor LV contractility (protocol in ). LV Systolic responses are shown in 7A-C : (A) Plots of left ventricular (LV) systolic developed pressure against balloon volume ; (B) LV maximal rate of tension development (+dP/dt) against balloon volume; (C) Maximal systolic elastance (E max ) – i.e., the slope from (A) ; (D) Plots of LV end diastolic pressure (EDP) against balloon volume; (E) Maximal rate of relaxation (-dP/dt) against balloon volume; (F) Stiffness, the reciprocal of the slope from (D) ; ( G) Percentage of LV contractile function recovery post-ischemia relative to baseline level. Data shown are mean ± S.E. N=4-8. *p<0.05, ***p<0.001, N=4-8 hearts/group. H) Blots of Cx43-pS368 (top) and total Cx43 (bottom) of LV samples infused with peptide for 20 minutes according to the protocol in . For hearts used in Western blots, the protocol did not proceed to the ischemia and reperfusion phases, being terminated after the peptide infusion step. Only those peptides competent to interact with Cx43 CT increase pS368 levels relative to total Cx43 above vehicle control.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: Langendorff I/R protocols were performed on adult mouse hearts instrumented to monitor LV contractility. Protocol in , except that a 20-minute peptide infusion was begun after ischemic injury at the initiation of reperfusion. (A) Plots of left ventricular (LV) developed pressure against balloon volume; (B) Maximal systolic elastance (E max ), the slope from (A); (C) Maximal rate of tension development (+dP/dt) against balloon volume; (D) Plots of end diastolic pressure (EDP) against balloon volume; (E) Stiffness, the reciprocal of the slope from (D); (F) Maximal rate of relaxation (-dP/dt) against balloon volume. * p<0.05, *** p<0.001, N=4-8. G) Laser scanning confocal microscopic fields from sections of Vehicle control, αCT1, and αCT11 group hearts stained for Cx43 (green), nuclei (DAPI-blue), and Alexa647-conjugated streptavidin (red). H) Average intensities of biotinylated peptide (indicated by streptavidin Alexa647 fluorescence intensity level relative to background) in Vehicle control, αCT1, and αCT11 groups. ** p<0.05; not significant (ns) N=5 hearts/group. Scale bar = 5 μm.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Staining, Fluorescence