Journal: PLoS ONE

Article Title: Low Density Lipoproteins Promote Unstable Calcium Handling Accompanied by Reduced SERCA2 and Connexin-40 Expression in Cardiomyocytes

doi: 10.1371/journal.pone.0058128

Figure Lengend Snippet: Effect of increasing doses of LDL (0, 50, 100 and 200 µg/mL) on connexin-40 (Cx40) and connexin-43 (Cx43) mRNA ( A ) or protein ( B ) levels in HL-1 myocyte cultures C Relationship between intracellular cholesteryl ester (CE) and Cx40 and Cx43 protein levels in HL-1 myocyte cultures. Results are shown as mean±SEM of three independent experiments performed in duplicate. *P<0.05 vs. cells incubated in absence of LDL.

Article Snippet: Gene expression of SERCA2, Cx40, and Cx43 was assessed by real time PCR-7000 Sequence Detection System of ABIPRISM (Applied Biosystems) using the following assays on demand: mouse Cx40 (Mn01264990_m1), mouse Cx43 (Mn00439105_m1), SERCA2 (Rn01499537_m1), RyR2 (Rn01470303_m1), IP3R-I (Rn01425720_m1), IP3R-III (Rn01470303_m1).

Techniques: Incubation

Journal: PLoS ONE

Article Title: Atrial Fibrillation-Linked Germline GJA5 /Connexin40 Mutants Showed an Increased Hemichannel Function

doi: 10.1371/journal.pone.0095125

Figure Lengend Snippet: ( A ) Representative fluorescent confocal images of Cx40-YFP, V85I-YFP and L221I-YFP (top panels). The overlaid fluorescent images on top of phase contrast images are also shown (bottom panels). All three constructs were able to form gap junction plaque-like structures at the cell-cell junction. Scale bar = 10 µm. ( B ) Voltage steps of 20 mV were applied to one cell of a transfected N2A cell pair and the junctional current (I j ) was recorded in the second cell. There was no significant difference between the I j in cell pairs expressing Cx40-YFP, V85I-YFP or L221I-YFP. The junctional conductance (G j ) was calculated and there was no significant difference between the G j of cell pairs expressing Cx40-YFP, V85I-YFP or L221I-YFP.

Article Snippet: Wild-type Cx40-YFP and YFP-tagged mutants were imaged using a Zeiss LSM 510-META confocal microscope as described earlier .

Techniques: Construct, Transfection, Expressing

Journal: PLoS ONE

Article Title: Atrial Fibrillation-Linked Germline GJA5 /Connexin40 Mutants Showed an Increased Hemichannel Function

doi: 10.1371/journal.pone.0095125

Figure Lengend Snippet: ( A ) Representative images of propidium iodide (PI)-uptake under divalent cation-free (DCF) conditions for isolated, individual, transfected HeLa cells. Successful transfection can be identified by their tagged green/yellow fluorescent proteins (green colour in the first column images). PI-uptake (red colour in the second column images) can be seen in cells expressing Cx26-GFP, V85I-YFP and L221I-YFP, but no uptake was seen in cells expressing YFP alone or Cx40-YFP. Scale bar = 20 µm. ( B ) Quantification of PI-uptake under DCF conditions. V85I-YFP (67.6%, n = 10) and L221I-YFP (83.2%, n = 10) showed a significant increase in PI-uptake compared to Cx40-YFP (4.6%, n = 14, ***indicates P<0.001). Other AF-linked Cx40 mutants, Q49X, L229M and I75F, were also studied and did not show any PI-uptake.

Article Snippet: Wild-type Cx40-YFP and YFP-tagged mutants were imaged using a Zeiss LSM 510-META confocal microscope as described earlier .

Techniques: Isolation, Transfection, Expressing

Journal: PLoS ONE

Article Title: Atrial Fibrillation-Linked Germline GJA5 /Connexin40 Mutants Showed an Increased Hemichannel Function

doi: 10.1371/journal.pone.0095125

Figure Lengend Snippet: ( A ) Comparison of PI-uptake for divalent cation containing (DCC) and divalent cation-free (DCF) conditions. The PI-uptake for cells expressing Cx26-GFP, V85I-YFP and L221I-YFP was significantly increased under DCF conditions compared to DCC conditions. Also the addition of the hemichannel blocker carbenoxolone (CBX, 100 µM) under DCF conditions significantly decreased PI-uptake. ( B ) [Ca 2+ ] o dose-dependent PI-uptake. Cx26-GFP, V85I-YFP and L221I-YFP all showed [Ca 2+ ] o dependent PI-uptake. Cx40-YFP did not show PI-uptake for any concentrations tested.

Article Snippet: Wild-type Cx40-YFP and YFP-tagged mutants were imaged using a Zeiss LSM 510-META confocal microscope as described earlier .

Techniques: Expressing

Journal: PLoS ONE

Article Title: Atrial Fibrillation-Linked Germline GJA5 /Connexin40 Mutants Showed an Increased Hemichannel Function

doi: 10.1371/journal.pone.0095125

Figure Lengend Snippet: ( A ) Representative confocal images showing the anti-Cx40 antibody localizations of untagged Cx40, V85I and L221I expressed in HeLa (top panels) and N2A (bottom panels) cells. In both cell lines, Cx40, V85I and L221I were all able to traffic to the cell-cell interface and form gap junction plaque-like structures. Scale bar = 10 µm. ( B ) There was no significant difference between the G j of cell pairs expressing Cx40, V85I or L221I. ( C ) Co-expression of Cx40V85I-IRES-GFP or L221I-IRES-GFP with Cx43-mRFP (V85I+Cx43 or L221I+Cx43) in N2A cell pairs showed a similar G j with cell pairs expressing Cx43-mRFP. The number of cell pairs are indicated on the bars.

Article Snippet: Wild-type Cx40-YFP and YFP-tagged mutants were imaged using a Zeiss LSM 510-META confocal microscope as described earlier .

Techniques: Expressing

Journal: PLoS ONE

Article Title: Atrial Fibrillation-Linked Germline GJA5 /Connexin40 Mutants Showed an Increased Hemichannel Function

doi: 10.1371/journal.pone.0095125

Figure Lengend Snippet: ( A ) Untagged V85I and L221I showed a significant increase in PI-uptake compared to both wild-type Cx40 and Cx43 under the divalent cation-free (DCF) conditions. ( B ) The addition of divalent cations (DCC, open bars) or CBX (100 µM, gray bars) blocked the PI-uptake from cells expressing Cx43, V85I and L221I. However, the addition of the pannexin 1 channel blocker probenecid (200 µM, hatched bars) did not affect PI-uptake. ( C ) PI-uptake in cells expressing untagged V85I or L221I under DCF were significantly (P<0.001 in both cases) reduced by the addition of flufenamic acid (FFA, 50 µM) or mefloquine (MFQ, 25 µM). The total number of experiments are indicated on the bar.

Article Snippet: Wild-type Cx40-YFP and YFP-tagged mutants were imaged using a Zeiss LSM 510-META confocal microscope as described earlier .

Techniques: Expressing

Journal: PLoS ONE

Article Title: Atrial Fibrillation-Linked Germline GJA5 /Connexin40 Mutants Showed an Increased Hemichannel Function

doi: 10.1371/journal.pone.0095125

Figure Lengend Snippet: ( A ) Representative confocal images of PI-uptake for HeLa cells transfected with wild-type Cx40 or Cx40 mutants (V85I or L221I). Time points of 0, 4 and 20 minutes of incubation with PI are displayed. During 20 minutes of incubation with PI, only cells expressing the V85I and L221I mutants showed PI uptake. Scale bar = 50 µm. ( B ) Ratio of current PI fluorescence intensity over the initial baseline fluorescence over a 20 minute incubation. L221I showed the fastest rate of PI-uptake, with V85I having a slightly slower rate of uptake, however, both L221I and V85I had similar levels of PI-uptake near the end of 20 minute incubation. The addition of 100 µM CBX blocked PI-uptake. Cx40 expressing cells failed to show any PI-uptake.

Article Snippet: Wild-type Cx40-YFP and YFP-tagged mutants were imaged using a Zeiss LSM 510-META confocal microscope as described earlier .

Techniques: Transfection, Incubation, Expressing, Fluorescence

Journal: PLoS ONE

Article Title: Atrial Fibrillation-Linked Germline GJA5 /Connexin40 Mutants Showed an Increased Hemichannel Function

doi: 10.1371/journal.pone.0095125

Figure Lengend Snippet: Voltage clamp ramp protocol (−40 to 110 mV) was used to study currents under divalent cation containing saline (DCC, black traces) and the divalent-cation free saline (DCF, red traces). Putative hemichannel currents (the current amplitude differences between red and black traces) were observed in cells expressing AF-linked Cx40 mutants, V85I (A), L221I (B) and wild-type Cx43 (C, left panel). Most of Cx40-expressing cells failed to show the current (C, right panel). The DCF-dependent currents in the mutant-expressing cells were largely blocked by carbenoxolone (CBX, 100 µM, green traces). Bar graph summarized the percentages of cells displayed hemichannel current during the voltage ramp under DCF conditions (D). The connexin constructs expressed and the numbers of cells recorded are indicated. Note only 4/33 cells expressing Cx40 displayed putative hemichannel current (data not shown).

Article Snippet: Wild-type Cx40-YFP and YFP-tagged mutants were imaged using a Zeiss LSM 510-META confocal microscope as described earlier .

Techniques: Expressing, Mutagenesis, Construct

Journal: PLoS ONE

Article Title: Atrial Fibrillation-Linked Germline GJA5 /Connexin40 Mutants Showed an Increased Hemichannel Function

doi: 10.1371/journal.pone.0095125

Figure Lengend Snippet: AF-linked germline Cx40 mutants have been shown to impair gap junction function via impaired localization (Q49X) or channel function (I75F). Dominant negative on Cx40 (Q49X and I75F) and/or transdominant negative actions on Cx43 were also observed (Q49X, I75F and L229M) , . Present study showed that AF-linked Cx40 mutants, V85I and L221I, increased hemichannel function.

Article Snippet: Wild-type Cx40-YFP and YFP-tagged mutants were imaged using a Zeiss LSM 510-META confocal microscope as described earlier .

Techniques: Dominant Negative Mutation

Journal: Oncotarget

Article Title: Connexin40 controls endothelial activation by dampening NFκB activation

doi: 10.18632/oncotarget.16438

Figure Lengend Snippet: (A) Representative en face images of eGFP in longitudinally opened carotids of Cx40 +/eGFP mice. eGFP (green) is highly expressed in the straight portions of the vessel (upper panel) but not at the iliac bifurcation (lower panel). DAPI (blue). (B) Cx40 expression (green) after modification of shear stress by a vascular cast in Cx40 +/eGFP mice. Shown are the contralateral undisturbed vessel (control) and the regions upstream (LLSS), within (HLSS) and downstream of the cast (OSS). Evans Blue (red). DAPI (blue). (C) Quantification of (B); N=8. (D) eGFP after modification of shear stress by a vascular cast in Cx40 +/eGFP mice. Shown are the contralateral undisturbed vessel (control) and the regions upstream (LLSS), within (HLSS) and downstream of the cast (OSS). (E) Cx40 expression in bEnd.3 cells exposed to static, HLSS, LLSS, OSS conditions for 24 hours was assessed by real-time qPCR. N=5. (F) Representative images of Cx40 expression (green) in bEnd.3 cells exposed to HLSS, LLSS, OSS for 24 hours. Scale bar represents 50 μm for in (A), (B) and (D), and 40 μm in (F).

Article Snippet: Subsequently, primary antibodies against Cx40 (Cx40-A; Alpha-Diagnostics lot #175455A8.6; 1/200), Cx37 (Cx37A11-A; Alpha-Diagnostics lot #175859A5-L; 1/50), NFκB (NFκB p65 (A) sc-109; Santa Cruz; 1/100) or Phospho-NFκB (Phospho-NFκB (S536)(93H1); Cell Signaling; 1/100) in blocking solution were applied overnight at 4°C.

Techniques: Expressing, Modification, Shear, Control

Journal: Oncotarget

Article Title: Connexin40 controls endothelial activation by dampening NFκB activation

doi: 10.18632/oncotarget.16438

Figure Lengend Snippet: (A) Cross-linking experiment with Cx40CT and peptides. Binding of IκBα-like or IκBα(5-16) peptides to Cx40CT was assessed after incubation with the chemical cross-linker BS 3 . All lanes show a band at ~16-17 kDa representing Cx40CT (arrow). The first and third lanes of the panel show an additional band at ~17-18kDa (arrow head), which is absent in lane 2, where the peptide is missing. (B) Representative en face images of PLA (out of 3 experiments) performed with antibodies targeting Cx40 and IκBα on rat carotid endothelium. Close proximity of Cx40 and IκBα (red) is observed in the intracellular compartment as well as at cell-cell contacts (left panel). Control assays revealed that the red staining observed was no longer observed after omitting either the Cx40 or the IκBα antibody from the PLA (right panel). Cx37 staining (green) is used to highlight the intercellular gap junctions. DAPI (blue). Scale bar represents 20 μm.

Article Snippet: Subsequently, primary antibodies against Cx40 (Cx40-A; Alpha-Diagnostics lot #175455A8.6; 1/200), Cx37 (Cx37A11-A; Alpha-Diagnostics lot #175859A5-L; 1/50), NFκB (NFκB p65 (A) sc-109; Santa Cruz; 1/100) or Phospho-NFκB (Phospho-NFκB (S536)(93H1); Cell Signaling; 1/100) in blocking solution were applied overnight at 4°C.

Techniques: Binding Assay, Incubation, Control, Staining

Journal: Oncotarget

Article Title: Connexin40 controls endothelial activation by dampening NFκB activation

doi: 10.18632/oncotarget.16438

Figure Lengend Snippet: (A) Lysates of bEnd.3 cells incubated or not with 10 ng/ml TNFα for 10, 15, 30 or 60 min were immunoblotted against NFκB, Cx40, IκBα, Phospho-IκBα or GAPDH. Whereas expression levels of NFκB, Cx40, and GAPDH were not affected by short-term stimulation with TNFα, the treatment induced phosphorylation and degradation of IκBα. (B) Quantification of (A) under control conditions or after incubation with 10 ng/ml TNFα for 15 min. N=3. (C) Expression of Cx40 (left panels) or NFκB (right panels; both in green) in control bEnd.3 cells and after 15 min stimulation with 10 ng/ml TNFα treated or not with siRNA for Cx40 or NT-siRNA. Note that in ECs in which Cx40 was silenced with siRNA (lower panels), NFκB translocation to the nucleus was enhanced after stimulation with TNFα. DAPI (blue). Scale bar represents 15 μm. (D) Cx40 expression in bEnd.3 cells exposed to Cx40 siRNA or NT-siRNA was assessed by real-time qPCR. N=3. (E) Expression of Cx40 (left) or Phospho-IκBα (right) in control bEnd.3 cells and after 15 min stimulation with 10 ng/ml TNFα treated with siRNA for Cx40 or NT-siRNA. N=3.

Article Snippet: Subsequently, primary antibodies against Cx40 (Cx40-A; Alpha-Diagnostics lot #175455A8.6; 1/200), Cx37 (Cx37A11-A; Alpha-Diagnostics lot #175859A5-L; 1/50), NFκB (NFκB p65 (A) sc-109; Santa Cruz; 1/100) or Phospho-NFκB (Phospho-NFκB (S536)(93H1); Cell Signaling; 1/100) in blocking solution were applied overnight at 4°C.

Techniques: Incubation, Expressing, Phospho-proteomics, Control, Translocation Assay

Journal: Oncotarget

Article Title: Connexin40 controls endothelial activation by dampening NFκB activation

doi: 10.18632/oncotarget.16438

Figure Lengend Snippet: (A) Cx40 immunostaining (green) in communication-incompetent HeLa cells (left panel) and in HeLa cells stably transfected with Cx40 (right panel). DAPI (blue). (B) Intercellular communication was measured by Lucifer Yellow microinjection during 3 min. Images are representative examples of Lucifer Yellow diffusion in parental HeLa cells (upper panel, N=6) and in HeLa cells stably transfected with Cx40 (lower panel, N=10). Asterisks indicate the microinjected cells. (C) Western blots (upper panel) showing the induction of Phospho-NFκB after 5 min stimulation with 20 ng/ml TNFα (+) as compared to control conditions (-) in parental HeLa cells and in HeLa cells stably transfected with Cx40. Expression of Cx40 reduced TNFα-induced NFκB phosphorylation (lower panel). N=3. (D) Cx40 immunostaining (green) in communication-incompetent HeLa cells transiently transfected with full-length Cx40 (left panel) or with Cx40CT (right panel). DAPI (blue). (E) Lysates of parental HeLa cells (lane 1) or transiently transfected with Cx40 (lanes 2 and 4) or Cx40CT (lanes 3 and 5) or stably transfected with Cx40 (lane 6) were immunoblotted against Cx40 and GAPDH. (F) Intercellular communication was measured by Lucifer Yellow microinjection during 5 min. Images are representative examples of Lucifer Yellow diffusion in HeLa cells transiently transfected with Cx40 (upper panel, N=8) or Cx40CT (lower panel, N=6). Asterisks indicate the microinjected cells. (G) Induction of Phospho-NFκB after 5 min stimulation with 20 ng/ml TNFα in HeLa cells transiently transfected with Cx40 or Cx40CT. Expression of Cx40 or Cx40CT revealed a similar protection against TNFα-induced NFκB phosphorylation. N=3. Scale bar represents 10 μm in (A) and (D), and 15 μm in (B) and (F).

Article Snippet: Subsequently, primary antibodies against Cx40 (Cx40-A; Alpha-Diagnostics lot #175455A8.6; 1/200), Cx37 (Cx37A11-A; Alpha-Diagnostics lot #175859A5-L; 1/50), NFκB (NFκB p65 (A) sc-109; Santa Cruz; 1/100) or Phospho-NFκB (Phospho-NFκB (S536)(93H1); Cell Signaling; 1/100) in blocking solution were applied overnight at 4°C.

Techniques: Immunostaining, Stable Transfection, Transfection, Microinjection, Diffusion-based Assay, Western Blot, Control, Expressing, Phospho-proteomics

Journal: Oncotarget

Article Title: Connexin40 controls endothelial activation by dampening NFκB activation

doi: 10.18632/oncotarget.16438

Figure Lengend Snippet: Representative images of SudanIV stainings are shown for the 3 flow regions of casted vessels (LLSS, HLSS, OSS) from Cx40 fl/fl Apoe -/- (A) and Tie2-cre Tg Cx40 fl/fl Apoe -/- (B, C) mice after 6 weeks of high-cholesterol diet. Intimal thickening was present in regions subjected to LLSS and OSS in Cx40 fl/fl Apoe -/- mice (A). Intimal thickening in response to LLSS and OSS was increased in Tie2-cre Tg Cx40 fl/fl Apoe -/- mice (B). The increased atherosclerotic response caused in half of the Tie2-cre Tg Cx40 fl/fl Apoe -/- mice a complete occlusion of the LLSS area that gave rise to atherosclerotic lesions within the cast (C). N=6-8 animals per group. Scale bar = 200 μm. (D, E) En face immunostaining for NFκB (in green) in carotid arteries of Cx40 fl/fl Apoe -/- (D) and Tie2-cre Tg Cx40 fl/fl Apoe -/- (E) mice. Note that NFκB signal was mostly cytoplasmic in Cx40 fl/fl Apoe -/- mice and more frequently localized to the nucleus in Tie2-cre Tg Cx40 fl/fl Apoe -/- mice. DAPI (blue). Scale bar represents 10 μm.

Article Snippet: Subsequently, primary antibodies against Cx40 (Cx40-A; Alpha-Diagnostics lot #175455A8.6; 1/200), Cx37 (Cx37A11-A; Alpha-Diagnostics lot #175859A5-L; 1/50), NFκB (NFκB p65 (A) sc-109; Santa Cruz; 1/100) or Phospho-NFκB (Phospho-NFκB (S536)(93H1); Cell Signaling; 1/100) in blocking solution were applied overnight at 4°C.

Techniques: Immunostaining

Journal: Frontiers in Physiology

Article Title: Low-Level Vagus Nerve Stimulation Reverses Obstructive Sleep Apnea-Related Atrial Fibrillation by Ameliorating Sympathetic Hyperactivity and Atrial Myocyte Injury

doi: 10.3389/fphys.2020.620655

Figure Lengend Snippet: (A) Representative examples of SNRT in the OSA group at baseline, the 1st hour, the 2nd hour, the 3rd hour, the 4th hour, and the 6th hour. (B) Changes in SNRT between the OSA and OSA+LLVS groups at each hour. (C) Changes in ion channels via gene expression level among the three groups. (D) Representative immunohistochemical staining of Cx 40 and Cx 43 on the LA among the three groups. (E) Quantitative analysis of Cx 43 and Cx 40-positive cells in the LA of each group. * p <0.05, ** p <0.01 vs. the control group; # p <0.05, ## p <0.01 vs. the OSA+LLVS group; SNRT, sinoatrial node recovery time; Cx 43, connexin 43; Cx40, connexin 40; H, hours.

Article Snippet: Connexin-40 (Cx40, LS-B959, 1:100, LifeSpan BioSciences, Seattle, WA, United States), and connexin-43 (Cx43, LS-B9771, 1:100, LifeSpan BioSciences, Seattle, WA, United States) were also detected, with quantitative analysis of integrated optical density (IOD) also conducted.

Techniques: Expressing, Immunohistochemical staining, Staining