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: Dentistry Journal

Article Title: Investigation of the Molecular Profile of Granular Cell Tumours and Schwannomas of the Oral Cavity

doi: 10.3390/dj10030038

Figure Lengend Snippet: Summary of the antibodies used for immunohistochemistry of GCTs and schwannomas.

Article Snippet: GAP43 , Bio-Techne Canada, Oakville On, NB300-143 , Rabbit polyclonal , Regenerating neural tissues/growth cones, GAP43 intracellular growth protein/membrane protein , 1/5000.

Techniques: Immunohistochemistry, Cell Surface Receptor Assay, Membrane, Plasmid Preparation

Journal: Neuroscience letters

Article Title: Overexpression of miR-338-5p in exosomes derived from mesenchymal stromal cells provides neuroprotective effects by the Cnr1/Rap1/Akt pathway after spinal cord injury in rats.

doi: 10.1016/j.neulet.2021.136124

Figure Lengend Snippet: Fig. 3. The expression of miR-338-5p was lower in the rats with SCI than the control rats, and the miR- 338-5p-overexpressing exosomes provided neuro protective effects after SCI. (A) Expression of miR- 338-5p was assessed by qRT-PCR at 1, 4 and 7 days after acute spinal cord injury in rats. (B, C) Western blot analysis of NF-M, GAP43, MAG and GFAP levels following various treatments at day 4 post-SCI. Data are presented as the mean ± SD. n = 4 per group (A), n = 3 per group (C), #p < 0.05, ##p < 0.01, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: After the membranes were blocked with 5% nonfat milk in TBST for 1 h at room temperature, they were incubated with primary antibodies overnight at 4◦C as follows: TSG101 (1:1000, Santa Cruz Biotechnology), HSP70 (1:1000, Santa Cruz Biotechnology), CD63 (1:1000, Santa Cruz Biotechnology), CD9 (1:1000, Santa Cruz Biotechnology), Cnr1 (1:1000, Santa Cruz Biotechnology), Rap1 (1:1000, Santa Cruz Biotechnology), neurofilament-M (NF-M) (1:1000, Proteintech Group), growth associated protein-43 (GAP43) (1:1000, Santa Cruz Biotechnology), myelinassociated glycoprotein (MAG) (1:1000, Proteintech Group), glial fibrillary acidic protein (GFAP) (1:1000, Proteintech Group), Bax (1:1000, Cell Signaling Technology), Bcl-2 (1:1000, Cell Signaling Technology), pro caspase-3 (1:1000, Cell Signaling Technology), cleaved-caspase-3 (1:1000, Cell Signaling Technology), total-PI3K (1:1000, Cell Signaling Technology), p-PI3K (1:1000, Cell Signaling Technology), total-AKT (1:1000, Cell Signaling Technology), and p-AKT (1:1000, Cell Signaling Technology).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot

Journal: Neuroscience letters

Article Title: Overexpression of miR-338-5p in exosomes derived from mesenchymal stromal cells provides neuroprotective effects by the Cnr1/Rap1/Akt pathway after spinal cord injury in rats.

doi: 10.1016/j.neulet.2021.136124

Figure Lengend Snippet: Fig. 4. Immunofluorescence double staining of NF-M, GAP43, MAG and GFAP levels following various treatments at day 4 post-SCI in rats. (A) Increased levels of the NF-M and GAP43 proteins in the region of anterior horn and adjacent lateral funiculus were observed in the miR-338-5p exo group compared to the other SCI groups at day 4 post-SCI. (B) Decreased protein levels of MAG and GFAP in the region of anterior horn and adjacent lateral funiculus were observed in the miR-338-5p exo group compared to the other SCI groups at day 4 post-SCI. Scale bar, 200 µm. (C) Semi-quantification of the mean fluorescence intensity of NF-M, GAP43, MAG and GFAP were measured. Data are presented as the mean ± SD. n = 3 per group, #p < 0.05, ##p < 0.01, ###p < 0.001, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: After the membranes were blocked with 5% nonfat milk in TBST for 1 h at room temperature, they were incubated with primary antibodies overnight at 4◦C as follows: TSG101 (1:1000, Santa Cruz Biotechnology), HSP70 (1:1000, Santa Cruz Biotechnology), CD63 (1:1000, Santa Cruz Biotechnology), CD9 (1:1000, Santa Cruz Biotechnology), Cnr1 (1:1000, Santa Cruz Biotechnology), Rap1 (1:1000, Santa Cruz Biotechnology), neurofilament-M (NF-M) (1:1000, Proteintech Group), growth associated protein-43 (GAP43) (1:1000, Santa Cruz Biotechnology), myelinassociated glycoprotein (MAG) (1:1000, Proteintech Group), glial fibrillary acidic protein (GFAP) (1:1000, Proteintech Group), Bax (1:1000, Cell Signaling Technology), Bcl-2 (1:1000, Cell Signaling Technology), pro caspase-3 (1:1000, Cell Signaling Technology), cleaved-caspase-3 (1:1000, Cell Signaling Technology), total-PI3K (1:1000, Cell Signaling Technology), p-PI3K (1:1000, Cell Signaling Technology), total-AKT (1:1000, Cell Signaling Technology), and p-AKT (1:1000, Cell Signaling Technology).

Techniques: Immunofluorescence, Double Staining, Fluorescence

Journal: Neuroscience letters

Article Title: Overexpression of miR-338-5p in exosomes derived from mesenchymal stromal cells provides neuroprotective effects by the Cnr1/Rap1/Akt pathway after spinal cord injury in rats.

doi: 10.1016/j.neulet.2021.136124

Figure Lengend Snippet: Fig. 6. Overexpression of miR-338-5p alleviated H2O2-induced cell injury in PC12 cells. (A) PC12 cells were transfected with NC mimics/inhibitors, miR-338-5p mimics and inhibitors. qRT-PCR analysis of the miR-338-5p level. (B, C) Measurement of intracellular ROS and SOD. (D) CCK-8 assay for cell viability. (E, F) Western blot analysis of NF-M, GAP43, MAG and GFAP levels following various treatments in PC12 cells. n = 3 per group, #p < 0.05, ##p < 0.01, ###p < 0.001, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: After the membranes were blocked with 5% nonfat milk in TBST for 1 h at room temperature, they were incubated with primary antibodies overnight at 4◦C as follows: TSG101 (1:1000, Santa Cruz Biotechnology), HSP70 (1:1000, Santa Cruz Biotechnology), CD63 (1:1000, Santa Cruz Biotechnology), CD9 (1:1000, Santa Cruz Biotechnology), Cnr1 (1:1000, Santa Cruz Biotechnology), Rap1 (1:1000, Santa Cruz Biotechnology), neurofilament-M (NF-M) (1:1000, Proteintech Group), growth associated protein-43 (GAP43) (1:1000, Santa Cruz Biotechnology), myelinassociated glycoprotein (MAG) (1:1000, Proteintech Group), glial fibrillary acidic protein (GFAP) (1:1000, Proteintech Group), Bax (1:1000, Cell Signaling Technology), Bcl-2 (1:1000, Cell Signaling Technology), pro caspase-3 (1:1000, Cell Signaling Technology), cleaved-caspase-3 (1:1000, Cell Signaling Technology), total-PI3K (1:1000, Cell Signaling Technology), p-PI3K (1:1000, Cell Signaling Technology), total-AKT (1:1000, Cell Signaling Technology), and p-AKT (1:1000, Cell Signaling Technology).

Techniques: Over Expression, Transfection, Quantitative RT-PCR, CCK-8 Assay, Western Blot

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Western blot showing expression of Cx43 in WT (lane 1) and in Cx43-null astrocytes transfected with full length Cx43 (lane 3), with Cx43 carboxyl terminus (lane 4) and with Cx43M257 (lane 5). Lane 2 corresponds to untransfected Cx43-null cells. Lanes 1–4 correspond to immunoblots performed with Cx43-18A antibody and lane 5 with Cx43-16A. (B) Time courses of calcein FRAP obtained from untreated (black squares) and 48 h carbenoxolone-treated (open triangles) WT astrocytes, and from untransfected (open circles) and transfected Cx43-null astrocytes with full length Cx43 (open squares) and Cx43M257 (black circles). Data were obtained from astrocytes cultured from a minimum of 3 litters of mice.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Western Blot, Expressing, Transfection, Cell Culture

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Dose-response curves obtained for untreated (black squares) and 48 h carbenoxolone-treated (open triangles) WT and untransfected Cx43-null spinal cord astrocytes exposed to the P2Y1R agonist 2-MeS-ATP. Note that blockade of gap junctional communication in WT astrocytes does not alter the half-maximal response (EC50 value) induced by the P2Y1R agonist. Mean values were obtained from four to seven independent experiments. (B) Dose-response curves obtained for WT (black squares), untransfected (open circles) and transfected Cx43-null astrocytes with full length Cx43 (open squares), Cx43M257 (black circles) and Cx43CT (black triangles). Note that both full length Cx43 and Cx43CT but not Cx43M257 shifted the EC50 values of 2-MeS-ATP obtained for Cx43-null astrocytes to those obtained in WT cells. Data were obtained from 5 to 8 litters.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Transfection

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: Bar histograms showing the mean values of P2Y1R expression levels in WT, untransfected Cx43-null (KO), and in Cx43-null transfected with Cx43 CT, Cx43 truncated at position 257 (M257) and with full length Cx43. An example of western blots for Cx43 and β-actin is shown above.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Expressing, Transfection, Western Blot

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Dose-response curves obtained for WT (black squares) and untransfected (open circles) and Cx43Δ260–280 transfected (black triangles) Cx43-null astrocytes exposed to 2-MeS-ATP, showing that deletion of the Cx43 SH3 domain does not rescue P2Y1 receptor function. Mean±SE values are from 100 to 200 cells obtained from three litters. (B) Dose-response curves obtained from WT (black squares), and Cx43-null astrocytes untreated (open circles) and treated (black triangles) with a membrane permeant peptide corresponding to amino acids 260–280 of Cx43CT. (C) Bar histograms of the mean±SE values of intracellular calcium mobilization induced by 100 nM 2-MeS-ATP recorded from WT, Cx43-null and Cx43-null transfected with Cx43CT and Cx43M257 in the absence and presence of 5 µM PP2. Note that only untransfected and Cx43M257 transfected Cx43-null astrocytes that were not exposed to PP2 did not respond to agonist with intracellular calcium levels similar to WT astrocytes. Mean ± SE values are from 4 litters (**P < 0.001; ANOVA followed by Newman-Keuls’ Multiple Comparison Test).

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Transfection

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Western blot showing decreased expression levels of Cx43 following exposure of spinal cord astrocytes to IL-1β (B) Dose-response curves obtained for 2-MeS-ATP performed on Fura-2 loaded WT and Cx43 KO astrocytes treated for 24 h with IL-1β (20 ng/mL). Note that exposure to the cytokine altered the agonist EC50 values in WT astrocytes. About 180 cells from three independent experiments were used in each condition.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Western Blot, Expressing

Journal: International journal of molecular medicine

Article Title: Secretome of EMSCs neutralizes LPS‑induced acute lung injury via aerosol administration.

doi: 10.3892/ijmm.2023.5307

Figure Lengend Snippet: Figure 1. Morphology and fluorescence identification of EMSCs. (A) EMSCs in P0 and P3. (B) Expression of MSC markers (CD44, Vimentin) and neural crest‑related biomarkers (Cx43, Sox9). EMSCs, ectodermal mesenchymal stem cells; Sox9, SRY‑related high‑mobility group box‑containing protein 9; Cx43, Connexin43.

Article Snippet: Briefly, samples in 24‐well plates were incubated with primary antibodies for CD44 (1:100, Boster, A00052), Cx43 (1:100, Boster, BA1727), Sox9 (1:100, Boster, PA1026‐1), Vimentin (1:100, Boster, PB9359) after being blocked with BSA.

Techniques: Fluorescence, Expressing