cx43 (mouse-anti-human  (Thermo Fisher)

Journal: International Journal of Molecular Sciences

Article Title: The Involvement of Cx43 in JNK1/2-Mediated Endothelial Mechanotransduction and Human Plaque Progression

doi: 10.3390/ijms24021174

Figure Lengend Snippet: JNK activation in inflammatory- and mechano-stimulated ECs is associated with the induction of Cx43 and proapoptotic caspases. ( A – F ) HUVECs were seeded into bifurcation flow-through cell culture slides, creating regions of laminar and non-uniform SS. Cells were exposed to SS by application of flow for 20 h. For the last 2 h, cells were stimulated with TNF-α with or without addition of JNK1/2 inhibitor SP600125. Flow was stopped and RNA was isolated from HUVECs and reverse transcribed into cDNA to perform RT-qPCR. Transcription levels are illustrated as x-fold mRNA compared with control treatment, which was set to 100%. Under SS conditions, TNF-α induced expression of JNK-associated transcription factors ( A , B ), of gap junction protein Cx43 ( C ) and the proapoptotic proteases caspase 9 ( E ) and caspase 3 ( F ), the latest was suppressed by JNK1/2 inhibition. No significant changes were observed for Cx40 ( D ). Data are presented as mean ± SEM, n = 8, analyzed by unpaired t-test ( A – E ) or one-way ANOVA followed by Tukey’s multiple comparison ( F ). ( G ) Linear regression analyses show a correlation of c-Jun and Cx43 transcription levels. n = 15 ( H – J ) Flow experiments were performed as described and protein expression of Cx43 was quantified using immunofluorescence. ( H ) Cx43 protein expression was higher in TNF-α treated HUVECs exposed to non-uniform SS compared to static conditions. ( I ) Increase in Cx43 protein expression in non-uniform SS-exposed HUVECs following TNF-α stimulation was suppressed by JNK1/2 inhibition. Data are presented as mean ± SEM, dots represent values from individual experiments, n = 7–9, analyzed by Kruskal–Wallis test followed by Dunn’s multiple comparison. ( J ) Exemplary microscopic images of Cx43 expression under non-uniform SS are shown (DAPI = blue, Cx43 = green, Cx40 = red; bar represents 50 µm). * p < 0.05, ** p < 0.01.

Article Snippet: Additionally, cross-sections were immunohistochemically stained for CD68 (mouse-anti-human, ready to use, DAKO, Hamburg, Germany) to detect plaque migrated macrophages, CD3 (mouse-anti-human, DAKO, 1:50) to detect T cells and Cx43 (mouse-anti-human, Invitrogen, 1:100) using CSA-Kit (DAKO), according to the manufacturer’s instructions.

Techniques: Activation Assay, Cell Culture, Isolation, Quantitative RT-PCR, Expressing, Inhibition, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: The Involvement of Cx43 in JNK1/2-Mediated Endothelial Mechanotransduction and Human Plaque Progression

doi: 10.3390/ijms24021174

Figure Lengend Snippet: Mechanic and inflammatory induction of Cx expression in HUVECs. HUVECs were seeded into laminar ( A – D ) or bifurcation flow-through cell culture slides ( E , F ) and exposed to SS by application of flow for 0–24 h. ( A , B ) RNA was isolated from HUVECs following 24 h flow or static conditions for RT-qPCR, showing higher transcription levels of both Cx43 ( A ) and Cx40 ( B ) following laminar SS compared with static conditions ( n = 6). ( C , D ) After 24 h of flow or static conditions, HUVECs were stained for Cx43 and Cx40 by immunofluorescence ( n = 9). Cx43 expression was significantly higher under laminar SS compared with static conditions ( C ), while no significant changes were observed for Cx40 ( D ). Data are presented using box and whisker plot, showing median, the interquartile range and minimum and maximum values. Wilcoxon matched-pairs signed rank test ( A , D ) or paired t test ( B , C ) was used for analysis. ( E , F ) At different time points (0 h, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h of flow) HUVECs were stained for Cx43 and Cx40 by immunofluorescence ( n = 3–4). After 8 h exposure to laminar SS significantly higher expression levels of Cx43 were observed compared with 0 h ( E ), while no significant differences were observed for Cx40 ( F ). Data are presented as mean ± SEM. Unpaired t-test was used to analyze the different time points compared with 0 h. Exemplary microscopic images of immunofluorescent stained HUVECs are shown on the right (Cx43 = green, Cx40 = red, DAPI = blue, scale bar = 100 µm). ( G – L ) Impact of inflammatory stimulation on Cx expression in HUVECs exposed to static or flow conditions was analyzed by immunofluorescent stainings. Stimulation with TNF-α for 2 h had no effect on Cx43 and Cx40 expression under static conditions ( G , J ) or under laminar SS conditions ( H , K ). HUVECs exposed to non-uniform SS exerted higher Cx43 expression following TNF-α stimulation ( I ), while Cx40 expression levels were unchanged ( L ). Data are presented as mean ± SEM, dots represent values from individual experiments, n = 5–9, unpaired t -test was used for analysis. * p < 0.05, ** p < 0.01.

Article Snippet: Additionally, cross-sections were immunohistochemically stained for CD68 (mouse-anti-human, ready to use, DAKO, Hamburg, Germany) to detect plaque migrated macrophages, CD3 (mouse-anti-human, DAKO, 1:50) to detect T cells and Cx43 (mouse-anti-human, Invitrogen, 1:100) using CSA-Kit (DAKO), according to the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Isolation, Quantitative RT-PCR, Staining, Immunofluorescence, Whisker Assay

Journal: International Journal of Molecular Sciences

Article Title: The Involvement of Cx43 in JNK1/2-Mediated Endothelial Mechanotransduction and Human Plaque Progression

doi: 10.3390/ijms24021174

Figure Lengend Snippet: Cx43 and caspase 3 induction are involved in proatherogenic cell responses of HUVECs exposed to non-uniform SS. Cell adhesion assay was performed following 20 h of SS exposure. After 18 h of perfusion with medium, HUVECs were perfused with medium, TNF-α or TNF-α together with an inhibitor of Cx43 (GAP19) or of JNK1/2 (SP600125) or a caspase inhibitor (CI) for 2 h, followed by 1 h of perfusion with THP-1 monocytes. Cells were stained and microscopic images in regions of laminar SS (6 per slide) and non-uniform SS (8 per slide) were obtained. Mean numbers of adherent THP-1 cells per visual field were assessed in laminar and non-uniform SS regions ( A ) and compared to TNF-α treatment, which was set to 100%. Comparable to SP600125, incubation with GAP19 or CI together with TNF-α resulted in lower adhesion of THP-1 cells to the HUVEC monolayer than sole TNF-α. Data are presented as mean ± SEM, dots represent values from individual experiments, n = 6–7, analyzed by one-way ANOVA followed by Sidak’s multiple comparison. ( B ) Shown are exemplary microscopic images of monocytes (blue arrow) adhered to the endothelial cell monolayer under nun-uniform SS. Scale bar = 100 µm. ( C ) Immunofluorescent staining of Cx43 and Cx40 was performed following adhesion assay and fluorescence microscopic images were obtained in regions of non-uniform SS. Exemplary images show THP-1 monocytes (violet), which have adhered to the endothelium (DAPI = blue, Cx43 = green, Cx40 = red; bar represents 50 µm). ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Additionally, cross-sections were immunohistochemically stained for CD68 (mouse-anti-human, ready to use, DAKO, Hamburg, Germany) to detect plaque migrated macrophages, CD3 (mouse-anti-human, DAKO, 1:50) to detect T cells and Cx43 (mouse-anti-human, Invitrogen, 1:100) using CSA-Kit (DAKO), according to the manufacturer’s instructions.

Techniques: Cell Adhesion Assay, Staining, Incubation, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: The Involvement of Cx43 in JNK1/2-Mediated Endothelial Mechanotransduction and Human Plaque Progression

doi: 10.3390/ijms24021174

Figure Lengend Snippet: Role of Cx43 in clinical manifestation. ( A – C ) Transcription levels of TNF-α ( A ) and the chemokine receptors CXCR4 ( B ) and CCR5 ( C ) in stable and vulnerable plaques, n = 14–20. ( D – G ) Numbers of infiltrated CD68-expressing macrophages in the PS ( D ) and the FC ( F ), n = 20. Exemplary microscopic images of CD68-staining are shown of the PS I and the FC region ( G ). ( H , I ) Higher rate of CD31-stained neovessels in vulnerable compared with stable plaques ( H ), n = 20. Exemplary microscopic images, showing brown-stained endothelial cells of neovessels (scale bar = 100 µm) ( I ). (J , K ) Increased numbers of Cx43-expressing cells in the PS region ( J ) and the FC ( K ) of vulnerable compared with stable plaques, n = 15–20. ( L ) Exemplary microscopic images show Cx43 expression by endothelial cells of neovessels (left upper image) and by macrophages in the inflammatory PS of a vulnerable plaque (right lower image), compared with Cx43-expressing cells in a stable plaque (right images). Data from gene plex assay and immunohistochemistry are presented as scatter dot plots showing mean ± SEM, dots represent values from individual experiments, unpaired t-test was used for analysis. ( M – P ) Spearman correlation analyses verify a correlation of Cx43 + cell counts with intraplaque TNF-α transcription, n = 35 ( M ), the rate of CD31 + neovessels, n = 36 ( N ), the mean area of the lipid core, n = 34 ( O ) and an inverse correlation with the FC thickness, n = 34 ( P ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Additionally, cross-sections were immunohistochemically stained for CD68 (mouse-anti-human, ready to use, DAKO, Hamburg, Germany) to detect plaque migrated macrophages, CD3 (mouse-anti-human, DAKO, 1:50) to detect T cells and Cx43 (mouse-anti-human, Invitrogen, 1:100) using CSA-Kit (DAKO), according to the manufacturer’s instructions.

Techniques: Expressing, Staining, Plex Assay, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: The Involvement of Cx43 in JNK1/2-Mediated Endothelial Mechanotransduction and Human Plaque Progression

doi: 10.3390/ijms24021174

Figure Lengend Snippet: Spearman correlation coefficient r of human plaque parameters.

Article Snippet: Additionally, cross-sections were immunohistochemically stained for CD68 (mouse-anti-human, ready to use, DAKO, Hamburg, Germany) to detect plaque migrated macrophages, CD3 (mouse-anti-human, DAKO, 1:50) to detect T cells and Cx43 (mouse-anti-human, Invitrogen, 1:100) using CSA-Kit (DAKO), according to the manufacturer’s instructions.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Involvement of Cx43 in JNK1/2-Mediated Endothelial Mechanotransduction and Human Plaque Progression

doi: 10.3390/ijms24021174

Figure Lengend Snippet: Qiagen QuantiTect ® Primer Assays used for RTq-PCR.

Article Snippet: Additionally, cross-sections were immunohistochemically stained for CD68 (mouse-anti-human, ready to use, DAKO, Hamburg, Germany) to detect plaque migrated macrophages, CD3 (mouse-anti-human, DAKO, 1:50) to detect T cells and Cx43 (mouse-anti-human, Invitrogen, 1:100) using CSA-Kit (DAKO), according to the manufacturer’s instructions.

Techniques:

Journal: Heliyon

Article Title: Differentiation of cardiomyocyte-like cells from human amniotic fluid mesenchymal stem cells by combined induction with human platelet lysate and 5-azacytidine

doi: 10.1016/j.heliyon.2020.e04844

Figure Lengend Snippet: The primers for RT–qPCR and products size.

Article Snippet: After fixation, the cells were blocked in 10% AB-serum in 1% BSA-PBS for 30 min at room temperature, and then incubated with mouse anti-human Cx43 primary antibodies (Sigma-aldrich, USA) for 2 h at 37 °C.

Techniques:

Journal: Heliyon

Article Title: Differentiation of cardiomyocyte-like cells from human amniotic fluid mesenchymal stem cells by combined induction with human platelet lysate and 5-azacytidine

doi: 10.1016/j.heliyon.2020.e04844

Figure Lengend Snippet: A: hPL ability to up-regulate cardiomyogenic specific genes expression. Each graph displays the expression levels of GATA4 , cTnT , Cx4 3 and Nkx2.5 , which were normalized to GAPDH and were relative to the control group. Data are presented as mean ± S.E. values. ∗ statistically significant versus control. # statistically significant between group. B: hPL combined with 5-aza ability to up-regulate cardiomyogenic specific genes expression. Each graph displays the expression levels of GATA4 , cTnT , Cx43 and Nkx2.5 , which were normalized to GAPDH and were relative to the control group. Data are presented as mean ± S.E. values. ∗ statistically significant versus control. # statistically significant between group.

Article Snippet: After fixation, the cells were blocked in 10% AB-serum in 1% BSA-PBS for 30 min at room temperature, and then incubated with mouse anti-human Cx43 primary antibodies (Sigma-aldrich, USA) for 2 h at 37 °C.

Techniques: Expressing

Journal: Heliyon

Article Title: Differentiation of cardiomyocyte-like cells from human amniotic fluid mesenchymal stem cells by combined induction with human platelet lysate and 5-azacytidine

doi: 10.1016/j.heliyon.2020.e04844

Figure Lengend Snippet: Ability of hPL to promote the differentiation of hAF-MSCs into cardiomyocyte-like cells. Each graph displays the expression levels of GATA4 , cTnT , Cx43 and Nkx2. 5 that were normalized to GAPDH and were relative to the control group. Data are presented as mean ± S.E. values. ∗ statistically significant versus control. # statistically significant between group.

Article Snippet: After fixation, the cells were blocked in 10% AB-serum in 1% BSA-PBS for 30 min at room temperature, and then incubated with mouse anti-human Cx43 primary antibodies (Sigma-aldrich, USA) for 2 h at 37 °C.

Techniques: Expressing

Journal: Heliyon

Article Title: Differentiation of cardiomyocyte-like cells from human amniotic fluid mesenchymal stem cells by combined induction with human platelet lysate and 5-azacytidine

doi: 10.1016/j.heliyon.2020.e04844

Figure Lengend Snippet: Detection of cardiomyogenic specific proteins; immunofluorescence staining FITC (495 nm/519 nm), green color for all cardiomyogenic specific proteins (A–I) and immunoenzymatic staining (J–L); GATA4 (localized in nucleus) staining (A) control group, (B) 10 μM 5-aza induced group, (C) 10 μM 5-aza with 20% hPL induced group; cTnT (localized in cytoplasm) staining (D) control group, (E) 10 μM 5-aza induced group, (F) 10 μM 5-aza 20% hPL induced group; Nkx2.5 (localized in nucleus) staining (G) control group, (H) 10 μM 5-aza induced group, (I) 10 μM 5-aza with 20% hPL induced group; Cx43 (localized in cell membrane) staining (J) control group, (K) 10 μM 5-aza induced group (black arrow), (L) 10 μM 5-aza with 20% hPL induced group (black arrow). (A-C and G-I) insets without nuclear counterstain showing no nuclear staining of two key core cardiac transcription factors. Scale bar = 100 μm.

Article Snippet: After fixation, the cells were blocked in 10% AB-serum in 1% BSA-PBS for 30 min at room temperature, and then incubated with mouse anti-human Cx43 primary antibodies (Sigma-aldrich, USA) for 2 h at 37 °C.

Techniques: Immunofluorescence, Staining

Journal: Heliyon

Article Title: Differentiation of cardiomyocyte-like cells from human amniotic fluid mesenchymal stem cells by combined induction with human platelet lysate and 5-azacytidine

doi: 10.1016/j.heliyon.2020.e04844

Figure Lengend Snippet: Image J analysis showing the results of CTCF (the expression levels of GATA4, cTnT, Nkx2.5 and Cx43 proteins signal). Data are presented as mean ± S.E. values. ∗ statistically significant versus control.

Article Snippet: After fixation, the cells were blocked in 10% AB-serum in 1% BSA-PBS for 30 min at room temperature, and then incubated with mouse anti-human Cx43 primary antibodies (Sigma-aldrich, USA) for 2 h at 37 °C.

Techniques: Expressing

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Reduced expressions of connexin 43 and VEGF in the first-trimester tissues from women with recurrent pregnancy loss

doi: 10.1186/s12958-016-0179-4

Figure Lengend Snippet: Sequences of primers used for qRT-PCR analysis

Article Snippet: Sections were incubated with appropriate blocking serum (VECTASTAIN UNIVERSAL ABC kit, VECTOR) at room temperature for 20 min followed by incubation with diluted mouse anti human Cx43 (Cell Signaling Technology) or rabbit anti human VEGF (Abcam) overnight in a humidified chamber at 4 °C.

Techniques:

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Reduced expressions of connexin 43 and VEGF in the first-trimester tissues from women with recurrent pregnancy loss

doi: 10.1186/s12958-016-0179-4

Figure Lengend Snippet: The expression of Cx43 in chorionic villi and decidua collected from RPL women and control group. a , b The protein expression of Cx43 was measured by immunohistochemistry (magnification X400) and evaluated by IRS scores. The control group exhibited a higher level of Cx43 protein compared to the RPL group. c , d The Cx43 protein expression was assessed by Western Blot and the band intensity was evaluated. The protein level of Cx43 is significantly higher in the control group than that in the RPL group. e The expression of Cx43 mRNA was reduced in both villi and decidua from the RPL group and those from the control group. Data were presented as mean ± standard deviation (SD). *: P < 0.05; ** P < 0.01; ***: P < 0.001

Article Snippet: Sections were incubated with appropriate blocking serum (VECTASTAIN UNIVERSAL ABC kit, VECTOR) at room temperature for 20 min followed by incubation with diluted mouse anti human Cx43 (Cell Signaling Technology) or rabbit anti human VEGF (Abcam) overnight in a humidified chamber at 4 °C.

Techniques: Expressing, Immunohistochemistry, Western Blot, Standard Deviation

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Reduced expressions of connexin 43 and VEGF in the first-trimester tissues from women with recurrent pregnancy loss

doi: 10.1186/s12958-016-0179-4

Figure Lengend Snippet: Relationship of Cx43 and VEGF expressed in the chorionic villi collected from RPL women versus controls. The protein levels of Cx43 and VEGF expressed in the chorionic villi were positively and linearly related as revealed by western blot in both RPL group ( a ) and control group ( b ), though the immunohistochemical result did not find any correlation in either group (c, d ). The same positive and linear relationship was detected in the mRNA expression in the RPL group ( e ), but not in the control group ( f )

Article Snippet: Sections were incubated with appropriate blocking serum (VECTASTAIN UNIVERSAL ABC kit, VECTOR) at room temperature for 20 min followed by incubation with diluted mouse anti human Cx43 (Cell Signaling Technology) or rabbit anti human VEGF (Abcam) overnight in a humidified chamber at 4 °C.

Techniques: Western Blot, Immunohistochemical staining, Expressing

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Reduced expressions of connexin 43 and VEGF in the first-trimester tissues from women with recurrent pregnancy loss

doi: 10.1186/s12958-016-0179-4

Figure Lengend Snippet: Relationship of Cx43 and VEGF expressed in the decidua collected from RPL women versus controls. In the decidua, there was no significant correlation of protein expression of Cx43 and VEGF from RPL women ( a , c ) or the control group ( b , d ). This is consistent with the results of mRNA expression in both groups ( e , f )

Article Snippet: Sections were incubated with appropriate blocking serum (VECTASTAIN UNIVERSAL ABC kit, VECTOR) at room temperature for 20 min followed by incubation with diluted mouse anti human Cx43 (Cell Signaling Technology) or rabbit anti human VEGF (Abcam) overnight in a humidified chamber at 4 °C.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Altered Expression Pattern of Molecular Factors Involved in Colonic Smooth Muscle Functions: An Immunohistochemical Study in Patients with Diverticular Disease

doi: 10.1371/journal.pone.0057023

Figure Lengend Snippet: Primary antibodies used in this study.

Article Snippet: Anti-human Cx43 , Mouse monoclonal , Santa Cruz Biotechnology, Santa Cruz, CA, USA , 1∶4500.

Techniques: