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: The Journal of Biological Chemistry

Article Title: A mouse model of inherited choline kinase β-deficiency presents with specific cardiac abnormalities and a predisposition to arrhythmia

doi: 10.1016/j.jbc.2022.101716

Figure Lengend Snippet: Reduced expression of cardiac conduction system markers in Chkb -deficient mice. RT–quantitative PCR analysis was used to monitor gene expression of atrial natriuretic peptide ( ANP ) ( A ), natriuretic peptide receptor-A ( NPRA ) ( B ), ventricular conduction system markers connexin 40 ( Cx40 ) ( C ), and hyperpolarization-activated cyclic nucleotide-gated channel-4 ( HCN4 ) ( D ). Expression levels were normalized to Gapdh via the ΔΔC T method. n = 3 to 6 mice per group, each bar represents mean ± SD, ∗ p < 0.01, ∗∗ p < 0.01; one-way ANOVA with Tukey’s multiple comparisons post hoc test. E and F , representative images and quantitation of cardiac muscle sections of 30-day-old Chkb +/+ , Chkb +/ − , and Chkb − / − mice stained with sarcomeric myosin MF20 ( green ) and Cx40 antibodies ( red ) along with a Bodipy nuclear stain. The scale bar represents 50 μM.

Article Snippet: After 1 h, blocking buffer solution was removed and replaced with blocking buffer containing primary antibodies for Connexin40 (Cx40) (1:50, Alpha Diagnostics, Cat# IGG1 CX40A), Sarcomeric myosin (MF20) (1:50, Developmental Studies Hybridoma Bank, Cat# MF-20).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitation Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: A mouse model of inherited choline kinase β-deficiency presents with specific cardiac abnormalities and a predisposition to arrhythmia

doi: 10.1016/j.jbc.2022.101716

Figure Lengend Snippet: Summary of cardiac events and their potential drivers due to Chkb deficiency. This study is the first to report that both heterozygous and homozygous Chkb (Choline kinase beta) deficiencies alter the cardiac lipid profile and membrane composition and are associated with cardiomyopathy. Chkb deficiency results in a significant decrease in the expression of ANP , its receptor NPRA , as well as ventricular conduction system markers (hyperpolarization-activated cyclic nucleotide-gated channel-4 [ HCN4 ] and connexin 40 [ Cx40 ]) in Chkb +/− and Chkb −/− mice. ANP expression has been shown to protect against the development of heart failure and is involved in the development of the embryonic ventricular conduction system. Defects in cardiac conduction system development in patients with congenital heart diseases can cause arrhythmias and may lead to sudden death. The decreased capacity of cardiac mitochondria from Chkb −/− mice to utilize fatty acids for oxygen production results in accumulation of AcCa in cardiac muscle. Increased levels of long-chain AcCa have been associated with cardiovascular disease risk, heart failure, left ventricle remodeling and function proportional to disease stage and severity. Furthermore, the alterations in specific cardiac signaling pathways in Chkb -deficient hearts (decreased p-AKT, p-GSKβ, and p-AMPK) lead to defective response to extracellular stimuli and render the hearts more susceptible to cardiomyopathy.

Article Snippet: After 1 h, blocking buffer solution was removed and replaced with blocking buffer containing primary antibodies for Connexin40 (Cx40) (1:50, Alpha Diagnostics, Cat# IGG1 CX40A), Sarcomeric myosin (MF20) (1:50, Developmental Studies Hybridoma Bank, Cat# MF-20).

Techniques: Expressing

Journal: Animal Cells and Systems

Article Title: Beneficial effect of simvastatin on human umbilical vein endothelial cells gap junctions induced by TNF-α

doi: 10.1080/19768354.2021.2023037

Figure Lengend Snippet: List of oligonucleotide primers used for real-time PCR analysis.

Article Snippet: Briefly, confluent cells grown on coverslips were fixed with 4% paraformaldehyde for 20 min, washed thoroughly with phosphate buffer saline (PBS), then blocked with 10% FBS and 0.3% Triton X-100 in PBS for 1 h. Next, the cells were incubated overnight at 4°C with the following antibodies: anti-Cx37 antibody (rabbit polyclonal Cx37 antibody raised against human; Cat. No. SAB4501180, Sigma-Aldrich), anti-Cx40 antibody (rabbit polyclonal Cx40 antibody raised against human; Cat. No. SAB1304973, Sigma-Aldrich) and anti-Cx43 antibody (rabbit polyclonal Cx43 antibody raised against human; Cat. No. C6219, Sigma-Aldrich).

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Animal Cells and Systems

Article Title: Beneficial effect of simvastatin on human umbilical vein endothelial cells gap junctions induced by TNF-α

doi: 10.1080/19768354.2021.2023037

Figure Lengend Snippet: Immunofluorescence results showing effect of simvastatin on GJIC targeting Cx37, Cx40 and Cx43. Cx37 (A) and Cx40 (B) expression was downregulated in gap junctions of HUVECs treated with TNF-α relative to the control group. Treatment of HUVECs with simvastatin upregulated Cx37 and Cx40 expression. Similarly, HUVECs treated with TNF-α exhibited Cx43 up-regulation (C) although simvastatin reversed this phenomenon. D: Quantitative comparison of FITC staining intensity normalized to control. n = 6, * P < 0.05, ** P < 0.01 vs. Control; # P < 0.05, ## P < 0.01 vs. TNF-α (+) Simvastain (−) group. Bar = 30 μm.

Article Snippet: Briefly, confluent cells grown on coverslips were fixed with 4% paraformaldehyde for 20 min, washed thoroughly with phosphate buffer saline (PBS), then blocked with 10% FBS and 0.3% Triton X-100 in PBS for 1 h. Next, the cells were incubated overnight at 4°C with the following antibodies: anti-Cx37 antibody (rabbit polyclonal Cx37 antibody raised against human; Cat. No. SAB4501180, Sigma-Aldrich), anti-Cx40 antibody (rabbit polyclonal Cx40 antibody raised against human; Cat. No. SAB1304973, Sigma-Aldrich) and anti-Cx43 antibody (rabbit polyclonal Cx43 antibody raised against human; Cat. No. C6219, Sigma-Aldrich).

Techniques: Immunofluorescence, Expressing, Staining

Journal: Animal Cells and Systems

Article Title: Beneficial effect of simvastatin on human umbilical vein endothelial cells gap junctions induced by TNF-α

doi: 10.1080/19768354.2021.2023037

Figure Lengend Snippet: Differential expression of Cx37, Cx40 and Cx43 mRNAs in HUVECs across groups. Cx37 and Cx40 had a 2-fold downregulation, whereas Cx43 was upregulated relative to the control group. Cx37 and Cx40 were significantly upregulated in HUVECs treated with TNF-α in combination with simvastatin relative to those treated with TNF-α alone. Cx43 was significantly downregulated. Data presented are means ± SEM, n = 6, ** P < 0.01 versus only TNF-α treatment group.

Article Snippet: Briefly, confluent cells grown on coverslips were fixed with 4% paraformaldehyde for 20 min, washed thoroughly with phosphate buffer saline (PBS), then blocked with 10% FBS and 0.3% Triton X-100 in PBS for 1 h. Next, the cells were incubated overnight at 4°C with the following antibodies: anti-Cx37 antibody (rabbit polyclonal Cx37 antibody raised against human; Cat. No. SAB4501180, Sigma-Aldrich), anti-Cx40 antibody (rabbit polyclonal Cx40 antibody raised against human; Cat. No. SAB1304973, Sigma-Aldrich) and anti-Cx43 antibody (rabbit polyclonal Cx43 antibody raised against human; Cat. No. C6219, Sigma-Aldrich).

Techniques: Expressing

Journal: Animal Cells and Systems

Article Title: Beneficial effect of simvastatin on human umbilical vein endothelial cells gap junctions induced by TNF-α

doi: 10.1080/19768354.2021.2023037

Figure Lengend Snippet: Cx37, Cx40 and Cx43 proteins were differentially expressed in HUVECs in different treatment groups. A: Western blots. 1: Control, 2: TNF-α (+)/Simvastatin (−), 3: TNF-α (+)/Simvastatin (+). B: qRT-PCR results showing relative transcript levels. Data presented are means ± SEM, n = 6, ** P < 0.01 versus only TNF-α treatment group.

Article Snippet: Briefly, confluent cells grown on coverslips were fixed with 4% paraformaldehyde for 20 min, washed thoroughly with phosphate buffer saline (PBS), then blocked with 10% FBS and 0.3% Triton X-100 in PBS for 1 h. Next, the cells were incubated overnight at 4°C with the following antibodies: anti-Cx37 antibody (rabbit polyclonal Cx37 antibody raised against human; Cat. No. SAB4501180, Sigma-Aldrich), anti-Cx40 antibody (rabbit polyclonal Cx40 antibody raised against human; Cat. No. SAB1304973, Sigma-Aldrich) and anti-Cx43 antibody (rabbit polyclonal Cx43 antibody raised against human; Cat. No. C6219, Sigma-Aldrich).

Techniques: Western Blot, Quantitative RT-PCR