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Pannexin 1 and pannexin 3 channels regulate skeletal muscle myoblast proliferation and differentiation.

Langlois S, Xiang X, Young K, Cowan BJ, Penuela S, Cowan KN - J. Biol. Chem. (2014)

Bottom Line: Using HSMM, we found that Panx1 expression promotes this process, whereas it was impaired in the presence of probenecid or carbenoxolone.Reduction of its endogenous expression using two Panx3 shRNAs significantly inhibited HSMM proliferation without triggering their differentiation.In summary, our results demonstrate that Panx1 and Panx3 are co-expressed in human skeletal muscle myoblasts and play a pivotal role in dictating the proliferation and differentiation status of these cells.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Surgery, Division of Paediatric Surgery, University of Ottawa, Children's Hospital of Eastern Ontario, Ottawa, Ontario K1H 8L1, Canada, Apoptosis Research Center, Children's Hospital of Eastern Ontario, Ottawa, Ontario K1H 8L1, Canada.

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Treatment with alkaline phosphatase alters the electrophoretic motility of the ∼70-kDa immunoreactive species of Panx3.A, parental HEK293T cells and HEK293T transfected with Panx3 were solubilized with Triton X-100, cross-linked with DSP, separated by SDS-PAGE, and immunoblotted with anti-Panx3. After the cross-links were reversed by boiling the samples in the presence of DTT, only the ∼43-kDa (monomer) and ∼70-kDa immunoreactive species of Panx3 were still present. Lysates of HEK293T cells transfected with Panx3 (B) or HSMM (C) were treated with CIP and submitted to SDS-PAGE. Although the electrophoretic motility of the lower Mr form of Panx3 was not affected (B), its ∼70-kDa immunoreactive species migrated further (arrow) after treatment of HEK293T and HSMM lysates with CIP. Representative Western blots of three independent experiments are shown.
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Figure 4: Treatment with alkaline phosphatase alters the electrophoretic motility of the ∼70-kDa immunoreactive species of Panx3.A, parental HEK293T cells and HEK293T transfected with Panx3 were solubilized with Triton X-100, cross-linked with DSP, separated by SDS-PAGE, and immunoblotted with anti-Panx3. After the cross-links were reversed by boiling the samples in the presence of DTT, only the ∼43-kDa (monomer) and ∼70-kDa immunoreactive species of Panx3 were still present. Lysates of HEK293T cells transfected with Panx3 (B) or HSMM (C) were treated with CIP and submitted to SDS-PAGE. Although the electrophoretic motility of the lower Mr form of Panx3 was not affected (B), its ∼70-kDa immunoreactive species migrated further (arrow) after treatment of HEK293T and HSMM lysates with CIP. Representative Western blots of three independent experiments are shown.

Mentions: Because of its apparent Mr, the ∼70-kDa immunoreactive species of Panx3 could possibly correspond to a Panx3 dimer. However, heating in combination with denaturing agents such as SDS and β-mercaptoethanol with or without DTT could not reduce it to a lower Mr form (data not shown). To further explore the possibility of a Panx3 dimer, we used the homobifunctional, amine-reactive reagent DSP to cross-link Panx3 following a method that has been used to show the oligomeric state of Panx1 (5). Triton X-100-solubilized extracts of wild-type or HEK293T cells transfected with Panx3 (∼43 kDa) were treated with the amino-reactive reagent DSP. After treatment with DSP, multiple bands were observed including the Panx3 monomer, the ∼70-kDa immunoreactive species, and bands at ∼85 kDa and higher (Fig. 4A). When the samples were boiled in the presence of DTT to reverse the cross-links, only the Panx3 monomer (∼43 kDa) and its ∼70-kDa immunoreactive species were still observed, suggesting that this band does not correspond to a Panx3 dimer. However, one of the bands detected after incubation with DSP at around 85 kDa may correspond to a dimer based on its size and its disappearance after boiling.


Pannexin 1 and pannexin 3 channels regulate skeletal muscle myoblast proliferation and differentiation.

Langlois S, Xiang X, Young K, Cowan BJ, Penuela S, Cowan KN - J. Biol. Chem. (2014)

Treatment with alkaline phosphatase alters the electrophoretic motility of the ∼70-kDa immunoreactive species of Panx3.A, parental HEK293T cells and HEK293T transfected with Panx3 were solubilized with Triton X-100, cross-linked with DSP, separated by SDS-PAGE, and immunoblotted with anti-Panx3. After the cross-links were reversed by boiling the samples in the presence of DTT, only the ∼43-kDa (monomer) and ∼70-kDa immunoreactive species of Panx3 were still present. Lysates of HEK293T cells transfected with Panx3 (B) or HSMM (C) were treated with CIP and submitted to SDS-PAGE. Although the electrophoretic motility of the lower Mr form of Panx3 was not affected (B), its ∼70-kDa immunoreactive species migrated further (arrow) after treatment of HEK293T and HSMM lysates with CIP. Representative Western blots of three independent experiments are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215249&req=5

Figure 4: Treatment with alkaline phosphatase alters the electrophoretic motility of the ∼70-kDa immunoreactive species of Panx3.A, parental HEK293T cells and HEK293T transfected with Panx3 were solubilized with Triton X-100, cross-linked with DSP, separated by SDS-PAGE, and immunoblotted with anti-Panx3. After the cross-links were reversed by boiling the samples in the presence of DTT, only the ∼43-kDa (monomer) and ∼70-kDa immunoreactive species of Panx3 were still present. Lysates of HEK293T cells transfected with Panx3 (B) or HSMM (C) were treated with CIP and submitted to SDS-PAGE. Although the electrophoretic motility of the lower Mr form of Panx3 was not affected (B), its ∼70-kDa immunoreactive species migrated further (arrow) after treatment of HEK293T and HSMM lysates with CIP. Representative Western blots of three independent experiments are shown.
Mentions: Because of its apparent Mr, the ∼70-kDa immunoreactive species of Panx3 could possibly correspond to a Panx3 dimer. However, heating in combination with denaturing agents such as SDS and β-mercaptoethanol with or without DTT could not reduce it to a lower Mr form (data not shown). To further explore the possibility of a Panx3 dimer, we used the homobifunctional, amine-reactive reagent DSP to cross-link Panx3 following a method that has been used to show the oligomeric state of Panx1 (5). Triton X-100-solubilized extracts of wild-type or HEK293T cells transfected with Panx3 (∼43 kDa) were treated with the amino-reactive reagent DSP. After treatment with DSP, multiple bands were observed including the Panx3 monomer, the ∼70-kDa immunoreactive species, and bands at ∼85 kDa and higher (Fig. 4A). When the samples were boiled in the presence of DTT to reverse the cross-links, only the Panx3 monomer (∼43 kDa) and its ∼70-kDa immunoreactive species were still observed, suggesting that this band does not correspond to a Panx3 dimer. However, one of the bands detected after incubation with DSP at around 85 kDa may correspond to a dimer based on its size and its disappearance after boiling.

Bottom Line: Using HSMM, we found that Panx1 expression promotes this process, whereas it was impaired in the presence of probenecid or carbenoxolone.Reduction of its endogenous expression using two Panx3 shRNAs significantly inhibited HSMM proliferation without triggering their differentiation.In summary, our results demonstrate that Panx1 and Panx3 are co-expressed in human skeletal muscle myoblasts and play a pivotal role in dictating the proliferation and differentiation status of these cells.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Surgery, Division of Paediatric Surgery, University of Ottawa, Children's Hospital of Eastern Ontario, Ottawa, Ontario K1H 8L1, Canada, Apoptosis Research Center, Children's Hospital of Eastern Ontario, Ottawa, Ontario K1H 8L1, Canada.

Show MeSH
Related in: MedlinePlus