Research Paper Volume 13, Issue 4 pp 4895—4910

Cholecalciferol (vitamin D3) has a direct protective activity against interleukin 6-induced atrophy in C2C12 myotubes

VD3 does not stimulate protein synthesis but activates STAT3. (A) Protein synthesis upon 24 h of treatment with 20 ng/ml IL6 in the presence or absence of 100nM VD3 was evaluated by SUnSET assay. Incorporation of puromycin into newly synthesized proteins was visualized by Western blotting and normalized on total proteins detected by Ponceau staining (representative images on the left of the panel). Densitometry of three experiments is shown on the right part of the panel. (B) Relative phosphorylation of proteins of Akt pathway after 30 min treatment with 100 nM VD3 through protein array analysis. (C) Representative western blot (top) and densitometry (bottom) of phosphorylated STAT3Y705 levels in C2C12 myotubes treated with 100 nM VD3, 20ng/mL IL6, or their combination for the indicated times. (D) Western blot (left) and densitometry (right) of phosphorylated STAT3Y705 levels in C2C12 myotubes treated with VD3, IL6, or their combination for 24 h. Data are presented as the mean ± SEM of three independent experiments except for (D), in which data are presented as the mean ± SD of the independent experiments indicated on the blot. **P

Figure 2. VD3 does not stimulate protein synthesis but activates STAT3. (A) Protein synthesis upon 24 h of treatment with 20 ng/ml IL6 in the presence or absence of 100nM VD3 was evaluated by SUnSET assay. Incorporation of puromycin into newly synthesized proteins was visualized by Western blotting and normalized on total proteins detected by Ponceau staining (representative images on the left of the panel). Densitometry of three experiments is shown on the right part of the panel. (B) Relative phosphorylation of proteins of Akt pathway after 30 min treatment with 100 nM VD3 through protein array analysis. (C) Representative western blot (top) and densitometry (bottom) of phosphorylated STAT3Y705 levels in C2C12 myotubes treated with 100 nM VD3, 20ng/mL IL6, or their combination for the indicated times. (D) Western blot (left) and densitometry (right) of phosphorylated STAT3Y705 levels in C2C12 myotubes treated with VD3, IL6, or their combination for 24 h. Data are presented as the mean ± SEM of three independent experiments except for (D), in which data are presented as the mean ± SD of the independent experiments indicated on the blot. **P<0.01.