Journal: Advanced Healthcare Materials

Article Title: Glucocorticoids Alter Bone Microvascular Barrier via MAPK/Connexin43 Mechanisms

doi: 10.1002/adhm.202404302

Figure Lengend Snippet: Glucocorticoids control endothelial and osteoblasts interactions via Cx43. A) RNA was isolated from 3D devices in the presence of Hyd, Pred, and Psl after 18 days. The expression of cell‐cell adhesion genes including zonula occludens‐1 (ZO‐1), vascular endothelial cadherin (VE‐cad), and connexin43 (Cx43) was examined by RT‐qPCR. The transcript levels were normalized to 18S. N = 3. B) The protein expression of Cx43 in the presence of GCs was analyzed by immunoblotting. The below panel shows the quantification of Cx43 levels compared to control. N = 5. C) Representative images of immunofluorescent signals for Cx43 (green), IB4 (red), and DAPI (blue) in bone sections from a mouse implanted with Psl pellets for 60 days or corresponding placebo control. Scale bars, 20 µm and 5 µm (zoom in images); Red arrows indicate the presence of Cx43 in the cell membrane area. Quantitative analysis of the Cx43‐positive membrane intensity in sections from each mouse stain. n = 10. D) Immunostaining for Cx43 (red) for co‐culture system of ECs and EGFP‐labeled HOBs (green). The nuclei were stained with DAPI (blue). Membrane localization of Cx43 is indicated by yellow arrows. Scale bars, 20 µm. E) Representative endothelial integrity images on the devices treated with GAP19 (5 µ m ; 30 min) using fluorescence labeled 70 kDa dextran. Scale bars, 100 µm. The below graph demonstrates the endothelial leakiness by diffusive permeability coefficient ( P d ) for control and GAP19 treated. The quantitative data are expressed as means ± SD. N = 3, n = 6; significant differences: *, p‐value < 0.05, * *, p‐value < 0.01, ** *, p‐value < 0.001.

Article Snippet: For Cx43 staining, the primary antibody against rabbit anti‐human Cx43 (1:100 dilution; Cell Signaling Technology) was incubated overnight at 4 °C.

Techniques: Control, Isolation, Expressing, Quantitative RT-PCR, Western Blot, Membrane, Staining, Immunostaining, Co-Culture Assay, Labeling, Fluorescence, Permeability

Journal: Advanced Healthcare Materials

Article Title: Glucocorticoids Alter Bone Microvascular Barrier via MAPK/Connexin43 Mechanisms

doi: 10.1002/adhm.202404302

Figure Lengend Snippet: Human osteoblast cells enhance endothelial barrier function via Cx43. A) Representative images showing endothelial integrity on the devices, using fluorescence‐labeled 70 kDa dextran. Scale bar, 100 µm. B) Graph demonstrates the endothelial leakiness measured by diffusive permeability coefficient ( P d ) for only HOBs, HOBs+ECs, and ECs. At least N = 3. C) RNA was isolated in HOBs, HOBs+ECs, and only ECs from 3D matrix devices. The expression of cell‐cell adhesion genes such as ZO‐1, VE‐cad, and Cx43 was examined by RT‐qPCR. The transcript levels were normalized to 18S. N = 3. D) The protein expression of Cx43 was determined by immunoblotting from genetically engineered HOBs; Control (Ctrl), Scramble CRISPR (Scramble), and Cx43 CRISPR (Cx43KO). β‐actin was used as a loading control. The right panel shows the quantification of Cx43 levels compared to Ctrl. N = 3. E) Representative images showing endothelial integrity of Ctrl, Scramble, and Cx43KO on the devices, using fluorescence labeled 70 kDa Texas red dextran. Scale bar, 100 µm. F) Graph demonstrates the endothelial leakiness measured by diffusive permeability coefficient ( P d ) for Ctrl, Scramble, and Cx43KO. N = 3. G) Representative images showing endothelial integrity of genetically engineered HOBs; Cx43 wild‐type (Cx43WT), phospho‐mimetic Cx43 Ser282 , Cx43SD, and blocking phospho‐mimetic Cx43 Ser282 , Cx43SA on the devices using fluorescence labeled 70 kDa Texas red dextran. Scale bar, 100 µm. H) Graph demonstrates the endothelial leakiness measured by diffusive permeability coefficient ( P d ) in Ctrl, Cx43WT, Cx43SD, and Cx43SA. N = 3, n = 6. I) Representative images showing barrier function in the devices seeded with ECs and genetically engineered HOBs expressing Cx43SA treated with Hyd, Pred, and Psl, using fluorescence labeled 70 kDa Texas red dextran. Scale bar, 100 µm. J) Graph demonstrates the endothelial leakiness measured by diffusive permeability coefficient ( P d ) in Cx43SA with Hyd, Pred, and Psl. The quantitative data are expressed as means ± SD. N = 3, n = 6; significant differences: *, p‐value < 0.05, * *, p‐value < 0.01.

Article Snippet: For Cx43 staining, the primary antibody against rabbit anti‐human Cx43 (1:100 dilution; Cell Signaling Technology) was incubated overnight at 4 °C.

Techniques: Fluorescence, Labeling, Permeability, Isolation, Expressing, Quantitative RT-PCR, Western Blot, Control, CRISPR, Blocking Assay

Journal: Advanced Healthcare Materials

Article Title: Glucocorticoids Alter Bone Microvascular Barrier via MAPK/Connexin43 Mechanisms

doi: 10.1002/adhm.202404302

Figure Lengend Snippet: Glucocorticoids regulate microvascular barrier function via MAPK/Cx43. A) Left, Western blot analysis showing GCs‐induced activation of Phospho‐Cx43 Ser282 , and Cx43 in co‐cultures of HOBs and ECs following stimulation with Hyd, Pred, and Psl for 30 min, 60 min, and 120 min. Inhibitor treatment was applied for 30 min. Right, Quantitative analysis of the Western blot results. N = 3. B) Left, Western blot analysis showing GCs‐induced activation of MAPKs (ERK, p38, and JNK) in co‐cultures of HOBs and ECs following stimulation with Hyd, Pred, and Psl for 30 min, 60 min, and 120 min. Inhibitor treatment was applied for 30 min. Right, Quantitative analysis of the Western blot results. N = 3. C) Representative images showing endothelial integrity in control and inhibitor‐treated group exposed to GCs on the devices for 30 min, using fluorescence‐labeled 70 kDa Texas Red dextran. Scale bar, 100 µm. Right panel, Graph demonstrates the endothelial leakiness measured by the diffusive permeability coefficient ( P d ) for control and inhibitor‐treated groups with GCs. The quantitative data are expressed as means ± SD. N = 3, n = 3; significant differences: *, p‐value < 0.05, * *, p‐value < 0.01, ** *, p‐value < 0.001.

Article Snippet: For Cx43 staining, the primary antibody against rabbit anti‐human Cx43 (1:100 dilution; Cell Signaling Technology) was incubated overnight at 4 °C.

Techniques: Western Blot, Activation Assay, Control, Fluorescence, Labeling, Permeability

Journal: Advanced Healthcare Materials

Article Title: Glucocorticoids Alter Bone Microvascular Barrier via MAPK/Connexin43 Mechanisms

doi: 10.1002/adhm.202404302

Figure Lengend Snippet: PCR primers used in this study.

Article Snippet: For Cx43 staining, the primary antibody against rabbit anti‐human Cx43 (1:100 dilution; Cell Signaling Technology) was incubated overnight at 4 °C.

Techniques:

Journal: Advanced healthcare materials

Article Title: Glucocorticoids Alter Bone Microvascular Barrier via MAPK/Connexin43 Mechanisms.

doi: 10.1002/adhm.202404302

Figure Lengend Snippet: Figure 4. Glucocorticoids control endothelial and osteoblasts interactions via Cx43. A) RNA was isolated from 3D devices in the presence of Hyd, Pred, and Psl after 18 days. The expression of cell-cell adhesion genes including zonula occludens-1 (ZO-1), vascular endothelial cadherin (VE-cad), and connexin43 (Cx43) was examined by RT-qPCR. The transcript levels were normalized to 18S. N = 3. B) The protein expression of Cx43 in the presence of GCs was analyzed by immunoblotting. The below panel shows the quantification of Cx43 levels compared to control. N = 5. C) Representative images of immunofluorescent signals for Cx43 (green), IB4 (red), and DAPI (blue) in bone sections from a mouse implanted with Psl pellets for 60 days or corresponding placebo control. Scale bars, 20 μm and 5 μm (zoom in images); Red arrows indicate the presence of Cx43 in the cell membrane area. Quantitative analysis of the Cx43-positive membrane intensity in sections from each mouse stain. n = 10. D) Immunostaining for Cx43 (red) for co-culture system of ECs and EGFP-labeled HOBs (green). The nuclei were stained with DAPI (blue). Membrane localization of Cx43 is indicated by yellow arrows. Scale bars, 20 μm. E) Representative endothelial integrity images on the devices treated with GAP19 (5 μm; 30 min) using fluorescence labeled 70 kDa dextran. Scale bars, 100 μm. The below graph demonstrates the endothelial leakiness by diffusive permeability coefficient (Pd) for control and GAP19 treated. The quantitative data are expressed as means ± SD. N = 3, n = 6; significant differences: *, p-value < 0.05, **, p-value < 0.01, ***, p-value < 0.001.

Article Snippet: For Cx43 staining, the primary antibody against rabbit anti-human Cx43 (1:100 dilution; Cell Signaling Technology) was incu- bated overnight at 4 °C.

Techniques: Control, Isolation, Expressing, Quantitative RT-PCR, Western Blot, Membrane, Staining, Immunostaining, Co-Culture Assay, Labeling, Permeability

Journal: Advanced healthcare materials

Article Title: Glucocorticoids Alter Bone Microvascular Barrier via MAPK/Connexin43 Mechanisms.

doi: 10.1002/adhm.202404302

Figure Lengend Snippet: Figure 5. Glucocorticoids regulate microvascular barrier function via MAPK/Cx43. A) Left, Western blot analysis showing GCs-induced activation of Phospho-Cx43Ser282, and Cx43 in co-cultures of HOBs and ECs following stimulation with Hyd, Pred, and Psl for 30 min, 60 min, and 120 min. Inhibitor treatment was applied for 30 min. Right, Quantitative analysis of the Western blot results. N = 3. B) Left, Western blot analysis showing GCs-induced activation of MAPKs (ERK, p38, and JNK) in co-cultures of HOBs and ECs following stimulation with Hyd, Pred, and Psl for 30 min, 60 min, and 120 min. Inhibitor treatment was applied for 30 min. Right, Quantitative analysis of the Western blot results. N = 3. C) Representative images showing endothelial integrity in control and inhibitor-treated group exposed to GCs on the devices for 30 min, using fluorescence-labeled 70 kDa Texas Red dextran. Scale bar, 100 μm. Right panel, Graph demonstrates the endothelial leakiness measured by the diffusive permeability coefficient (Pd) for control and inhibitor- treated groups with GCs. The quantitative data are expressed as means ± SD. N = 3, n = 3; significant differences: *, p-value < 0.05, **, p-value < 0.01, ***, p-value < 0.001.

Article Snippet: For Cx43 staining, the primary antibody against rabbit anti-human Cx43 (1:100 dilution; Cell Signaling Technology) was incu- bated overnight at 4 °C.

Techniques: Western Blot, Activation Assay, Control, Labeling, Permeability

Journal: Advanced healthcare materials

Article Title: Glucocorticoids Alter Bone Microvascular Barrier via MAPK/Connexin43 Mechanisms.

doi: 10.1002/adhm.202404302

Figure Lengend Snippet: Figure 4. Glucocorticoids control endothelial and osteoblasts interactions via Cx43. A) RNA was isolated from 3D devices in the presence of Hyd, Pred, and Psl after 18 days. The expression of cell-cell adhesion genes including zonula occludens-1 (ZO-1), vascular endothelial cadherin (VE-cad), and connexin43 (Cx43) was examined by RT-qPCR. The transcript levels were normalized to 18S. N = 3. B) The protein expression of Cx43 in the presence of GCs was analyzed by immunoblotting. The below panel shows the quantification of Cx43 levels compared to control. N = 5. C) Representative images of immunofluorescent signals for Cx43 (green), IB4 (red), and DAPI (blue) in bone sections from a mouse implanted with Psl pellets for 60 days or corresponding placebo control. Scale bars, 20 μm and 5 μm (zoom in images); Red arrows indicate the presence of Cx43 in the cell membrane area. Quantitative analysis of the Cx43-positive membrane intensity in sections from each mouse stain. n = 10. D) Immunostaining for Cx43 (red) for co-culture system of ECs and EGFP-labeled HOBs (green). The nuclei were stained with DAPI (blue). Membrane localization of Cx43 is indicated by yellow arrows. Scale bars, 20 μm. E) Representative endothelial integrity images on the devices treated with GAP19 (5 μm; 30 min) using fluorescence labeled 70 kDa dextran. Scale bars, 100 μm. The below graph demonstrates the endothelial leakiness by diffusive permeability coefficient (Pd) for control and GAP19 treated. The quantitative data are expressed as means ± SD. N = 3, n = 6; significant differences: *, p-value < 0.05, **, p-value < 0.01, ***, p-value < 0.001.

Article Snippet: Primary antibody against rabbit anti-human Cx43 (1:100 dilution in blocking solution; Cell Signaling Technology) was incubated overnight at 4 °C.

Techniques: Control, Isolation, Expressing, Quantitative RT-PCR, Western Blot, Membrane, Staining, Immunostaining, Co-Culture Assay, Labeling, Permeability

Journal: Advanced healthcare materials

Article Title: Glucocorticoids Alter Bone Microvascular Barrier via MAPK/Connexin43 Mechanisms.

doi: 10.1002/adhm.202404302

Figure Lengend Snippet: Figure 5. Glucocorticoids regulate microvascular barrier function via MAPK/Cx43. A) Left, Western blot analysis showing GCs-induced activation of Phospho-Cx43Ser282, and Cx43 in co-cultures of HOBs and ECs following stimulation with Hyd, Pred, and Psl for 30 min, 60 min, and 120 min. Inhibitor treatment was applied for 30 min. Right, Quantitative analysis of the Western blot results. N = 3. B) Left, Western blot analysis showing GCs-induced activation of MAPKs (ERK, p38, and JNK) in co-cultures of HOBs and ECs following stimulation with Hyd, Pred, and Psl for 30 min, 60 min, and 120 min. Inhibitor treatment was applied for 30 min. Right, Quantitative analysis of the Western blot results. N = 3. C) Representative images showing endothelial integrity in control and inhibitor-treated group exposed to GCs on the devices for 30 min, using fluorescence-labeled 70 kDa Texas Red dextran. Scale bar, 100 μm. Right panel, Graph demonstrates the endothelial leakiness measured by the diffusive permeability coefficient (Pd) for control and inhibitor- treated groups with GCs. The quantitative data are expressed as means ± SD. N = 3, n = 3; significant differences: *, p-value < 0.05, **, p-value < 0.01, ***, p-value < 0.001.

Article Snippet: Primary antibody against rabbit anti-human Cx43 (1:100 dilution in blocking solution; Cell Signaling Technology) was incubated overnight at 4 °C.

Techniques: Western Blot, Activation Assay, Control, Labeling, Permeability

Journal: Medicina

Article Title: Mutations of the Cx43 Gene in Non-Small Cell Lung Cancer: Association with Aberrant Localization of Cx43 Protein Expression and Tumor Progression

doi: 10.3390/medicina60101641

Figure Lengend Snippet: Relationships between Cx43 protein expression and clinicopathological characteristics.

Article Snippet: A monoclonal anti-human Cx43 antibody (Zymed Laboratories, Inc., San Francisco, CA, USA, dilution 1:250) was used, with an incubation time of 60 min at room temperature.

Techniques: Expressing, Membrane, Immunohistochemistry

Journal: Medicina

Article Title: Mutations of the Cx43 Gene in Non-Small Cell Lung Cancer: Association with Aberrant Localization of Cx43 Protein Expression and Tumor Progression

doi: 10.3390/medicina60101641

Figure Lengend Snippet: Immunohistochemical analysis of Cx43 protein expression using a monoclonal Cx43 anti-human antibody on paraffin sections of lung tumor specimens. ( A ) Negative control showing no Cx43 immunostaining (×200). ( B ) Positive control showing Cx43 protein expressed in the membrane of non-tumor lung tissue (arrow) (×400). ( C ) Nuclear localization of Cx43 in tumor cells (arrow) (patient 888C) (×400). ( D ) Cx43 expression in the cytoplasm of tumor cells (arrow) (patient 245D) (×400).

Article Snippet: A monoclonal anti-human Cx43 antibody (Zymed Laboratories, Inc., San Francisco, CA, USA, dilution 1:250) was used, with an incubation time of 60 min at room temperature.

Techniques: Immunohistochemical staining, Expressing, Negative Control, Immunostaining, Positive Control, Membrane

Journal: Medicina

Article Title: Mutations of the Cx43 Gene in Non-Small Cell Lung Cancer: Association with Aberrant Localization of Cx43 Protein Expression and Tumor Progression

doi: 10.3390/medicina60101641

Figure Lengend Snippet: Cx43 DNA sequencing results of 15 patients with Cx43 positive and/or negative immunostaining and their clinicopathological characteristic.

Article Snippet: A monoclonal anti-human Cx43 antibody (Zymed Laboratories, Inc., San Francisco, CA, USA, dilution 1:250) was used, with an incubation time of 60 min at room temperature.

Techniques: DNA Sequencing, Immunostaining, Sequencing, Mutagenesis

Journal: Medicina

Article Title: Mutations of the Cx43 Gene in Non-Small Cell Lung Cancer: Association with Aberrant Localization of Cx43 Protein Expression and Tumor Progression

doi: 10.3390/medicina60101641

Figure Lengend Snippet: Growth curves of CL-3 lung cancer cells expressing wild-type or mutant Cx43. Cells were transfected with wild-type Cx43 , vector control, or mutant Cx43 constructs (Cx43-18,19 and Cx43-57). Cell numbers were counted daily for 7 days. Data points represent the mean of three independent experiments + standard error of the mean.

Article Snippet: A monoclonal anti-human Cx43 antibody (Zymed Laboratories, Inc., San Francisco, CA, USA, dilution 1:250) was used, with an incubation time of 60 min at room temperature.

Techniques: Expressing, Mutagenesis, Transfection, Plasmid Preparation, Control, Construct

Journal: Medicina

Article Title: Mutations of the Cx43 Gene in Non-Small Cell Lung Cancer: Association with Aberrant Localization of Cx43 Protein Expression and Tumor Progression

doi: 10.3390/medicina60101641

Figure Lengend Snippet: Kaplan–Meier survival curves for NSCLC patients stratified by Cx43 protein localization. Three groups are represented: patients with Cx43 membrane expression and negative immunostaining (black line), patients with Cx43 positive staining in the cytoplasm (dotted line), and patients with Cx43 positive staining in the nucleus (grey line). The x -axis represents days after surgery, while the y -axis shows the survival percentage.

Article Snippet: A monoclonal anti-human Cx43 antibody (Zymed Laboratories, Inc., San Francisco, CA, USA, dilution 1:250) was used, with an incubation time of 60 min at room temperature.

Techniques: Membrane, Expressing, Immunostaining, Staining