Altered ERK/AKT signaling in <i>Ano6</i>-KD C2C12 myoblasts.

<p>(A) Western blotting analysis of ERK phosphorylation (pERK) and total ERK expression (ERK) in C2C12 myoblasts of different stable lines. Three independent experiments per cell line were loaded on the gel. (B) Normalized expression levels of ERK phosphorylation and total ERK by membrane densitometry. (C) Western blotting analysis of AKT phosphorylation (pAKT) and total AKT expression (AKT) in C2C12 myoblasts of different stable lines. Three independent experiments per cell line were loaded on the gel. (D) Normalized expression levels of AKT phosphorylation and total AKT by membrane densitometry. (E) Western blotting analysis of cyclin D1 in C2C12 myoblasts of different stable lines. (F) Normalized expression levels of cyclin D1 by membrane densitometry. Three independent experiments per cell line were loaded on the gel. GAPDH was used as a loading control. *p<0.05.</p

Effects of <i>Ano6</i>-KD on the proliferation of C2C12 myoblasts.

<p>(A) Relative expression of Ano6 (normalized to GAPDH) examined by quantitative RT-PCR in C2C12 stable cells lines (Scramble [shSCR], <i>Ano6</i>-KD). (B) Representative photographs of stable C2C12 cell lines expressing either a Scramble shRNA or the shRNA targeting <i>Ano6</i>-KD 48 hours post plating. (C) Quantitative analysis of C2C12 myoblast proliferation using the MTT assay. Scale bar  =  150 μm. ***p<0.001.</p

Anoctamin 6 Regulates C2C12 Myoblast Proliferation

<div><p>Anoctamin 6 (<i>Ano6</i>) belongs to a conserved gene family (TMEM16) predicted to code for eight transmembrane proteins with putative Ca²⁺-activated chloride channel (CaCC) activity. Recent work revealed that disruption of <i>ANO6</i> leads to a blood coagulation defect and impaired skeletal development. However, its function in skeletal muscle cells remains to be determined. By using a RNA interference mediated (RNAi) loss-of-function approach, we show that <i>Ano6</i> regulates C2C12 myoblast proliferation. <i>Ano6</i> is highly expressed in C2C12 myoblasts and its expression decreases upon differentiation. Knocking down <i>Ano6</i> significantly reduces C2C12 myoblast proliferation but has minimal effect on differentiation. <i>Ano6</i> deficiency significantly reduces ERK/AKT phosphorylation, which has been shown to be involved in regulation of cancer cell proliferation by another Anoctamin member. Taken together, our data demonstrate for the first time that <i>Ano6</i> plays an essential role in C2C12 myoblast proliferation, likely via regulating the ERK/AKT signaling pathway.</p></div

Expression of Ano6 in C2C12 muscle cells and mouse skeletal muscle.

<p>(A) Semi-quantitative RT-PCR analysis of Ano6 and GAPDH expression in C2C12 cells during differentiation. (B) Relative expression of Ano6 (normalized to GAPDH) examined by qRT-PCR in C2C12 cells during differentiation. (C) Relative expression of Ano6 (normalized to GAPDH) examined by qRT-PCR in the quadriceps muscles of mice at 6 days, 6 weeks and 6 months of age. **p<0.01; ***p<0.001.</p

Representative micrographs of Scramble and <i>Ano6</i>-KD C2C12 myotubes.

<p>(A) Scramble and <i>Ano6</i>-KD stable cells, which also express mCherry, were imaged 3days after differentiation. (B) Fusion index of Scramble and <i>Ano6</i>-KD stable C2C12 cells after differentiation for 3 days. Scale bar  =  150 μm. (C,D) Fold difference of myogenin and myosin heavy chain (MHC) expression as examined by RT-PCR of C2C12 cells during differentiation.</p

Effect of the ERK inhibitor UO126 on proliferation of control and <i>Ano6</i>-KD C2C12 myoblasts.

<p>(A) Proliferation analysis of control C2C12 cells treated by UO126 (10 μM) or the vehicle alone (DMSO) measured by MTT assay. (B) Proliferation analysis of <i>Ano6</i>-KD C2C12 cells treated by UO126 or the vehicle alone (DMSO) measured by MTT assay. Note that the dashed lines were re-plotted from panel A.</p