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The RNA-binding protein Staufen1 impairs myogenic differentiation via a c-myc-dependent mechanism.

Ravel-Chapuis A, Crawford TE, Blais-Crépeau ML, Bélanger G, Richer CT, Jasmin BJ - Mol. Biol. Cell (2014)

Bottom Line: Cells overexpressing Staufen1 differentiated poorly, as evidenced by reductions in the differentiation and fusion indices and decreases in MyoD, myogenin, MEF2A, and MEF2C, independently of Staufen-mediated mRNA decay.By contrast, the knockdown of Staufen1 decreased c-myc levels in myoblasts.Collectively our results show that Staufen1 is highly expressed during early stages of differentiation/development and that it can impair differentiation by regulating c-myc, thereby highlighting the multifunctional role of Staufen1 in skeletal muscle cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.

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Staufen1 overexpression inhibits myogenic differentiation. (A) Representative Western blots showing expression of Staufen1-HA in stable C2C12 cell lines. Two clones displaying transgene expression were chosen (#15 and #25), along with a control stable cell line. (B) Immunofluorescence of stable cell lines after 96 h of differentiation. Cells were stained with a pan-MyHC antibody (red) and nuclei with DAPI (blue). Scale bars, 100 um. (C) Fusion index (n = 5), (D) differentiation index (n = 5), and (E) myotube surface area (n = 3) of stable cell lines after 96 h of differentiation. Asterisks indicate significance (*p ≤ 0.05, **p ≤ 0.01).
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Figure 4: Staufen1 overexpression inhibits myogenic differentiation. (A) Representative Western blots showing expression of Staufen1-HA in stable C2C12 cell lines. Two clones displaying transgene expression were chosen (#15 and #25), along with a control stable cell line. (B) Immunofluorescence of stable cell lines after 96 h of differentiation. Cells were stained with a pan-MyHC antibody (red) and nuclei with DAPI (blue). Scale bars, 100 um. (C) Fusion index (n = 5), (D) differentiation index (n = 5), and (E) myotube surface area (n = 3) of stable cell lines after 96 h of differentiation. Asterisks indicate significance (*p ≤ 0.05, **p ≤ 0.01).

Mentions: To determine specifically the effect of Staufen1 in skeletal muscle cell development and investigate its involvement in myogenic control, we sought to prevent the decrease of Staufen1 by establishing stable C2C12 myoblast clones that overexpress a hemagglutinin (HA)-tagged Staufen1 construct. Stable C2C12 myoblasts were obtained by selecting for neomycin resistance with G418. Neomycin-resistant clones were then screened for Staufen1-HA expression by Western blotting using an anti-HA antibody (Figure 4A). Two clones, #15 and #25, were selected, along with a control cell line established with a pcDNA3 control vector. Comparative analysis between control and Staufen1-HA #25 was performed by Western blot using anti-Staufen1 antibodies and showed that Staufen1-HA is overexpressed by at least ∼40% in clone #25 (unpublished data).


The RNA-binding protein Staufen1 impairs myogenic differentiation via a c-myc-dependent mechanism.

Ravel-Chapuis A, Crawford TE, Blais-Crépeau ML, Bélanger G, Richer CT, Jasmin BJ - Mol. Biol. Cell (2014)

Staufen1 overexpression inhibits myogenic differentiation. (A) Representative Western blots showing expression of Staufen1-HA in stable C2C12 cell lines. Two clones displaying transgene expression were chosen (#15 and #25), along with a control stable cell line. (B) Immunofluorescence of stable cell lines after 96 h of differentiation. Cells were stained with a pan-MyHC antibody (red) and nuclei with DAPI (blue). Scale bars, 100 um. (C) Fusion index (n = 5), (D) differentiation index (n = 5), and (E) myotube surface area (n = 3) of stable cell lines after 96 h of differentiation. Asterisks indicate significance (*p ≤ 0.05, **p ≤ 0.01).
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Related In: Results  -  Collection

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Figure 4: Staufen1 overexpression inhibits myogenic differentiation. (A) Representative Western blots showing expression of Staufen1-HA in stable C2C12 cell lines. Two clones displaying transgene expression were chosen (#15 and #25), along with a control stable cell line. (B) Immunofluorescence of stable cell lines after 96 h of differentiation. Cells were stained with a pan-MyHC antibody (red) and nuclei with DAPI (blue). Scale bars, 100 um. (C) Fusion index (n = 5), (D) differentiation index (n = 5), and (E) myotube surface area (n = 3) of stable cell lines after 96 h of differentiation. Asterisks indicate significance (*p ≤ 0.05, **p ≤ 0.01).
Mentions: To determine specifically the effect of Staufen1 in skeletal muscle cell development and investigate its involvement in myogenic control, we sought to prevent the decrease of Staufen1 by establishing stable C2C12 myoblast clones that overexpress a hemagglutinin (HA)-tagged Staufen1 construct. Stable C2C12 myoblasts were obtained by selecting for neomycin resistance with G418. Neomycin-resistant clones were then screened for Staufen1-HA expression by Western blotting using an anti-HA antibody (Figure 4A). Two clones, #15 and #25, were selected, along with a control cell line established with a pcDNA3 control vector. Comparative analysis between control and Staufen1-HA #25 was performed by Western blot using anti-Staufen1 antibodies and showed that Staufen1-HA is overexpressed by at least ∼40% in clone #25 (unpublished data).

Bottom Line: Cells overexpressing Staufen1 differentiated poorly, as evidenced by reductions in the differentiation and fusion indices and decreases in MyoD, myogenin, MEF2A, and MEF2C, independently of Staufen-mediated mRNA decay.By contrast, the knockdown of Staufen1 decreased c-myc levels in myoblasts.Collectively our results show that Staufen1 is highly expressed during early stages of differentiation/development and that it can impair differentiation by regulating c-myc, thereby highlighting the multifunctional role of Staufen1 in skeletal muscle cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.

Show MeSH
Related in: MedlinePlus