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

VDR, but not RXR nor 1,25D3-MARRS (Pdia3), mediates VD3 anti-atrophic activity. (A) C2C12 myotubes were transfected with non-targeting or Vdr specific siRNAs. 24 h after silencing, C2C12 myotubes were treated with 20ng/mL IL6 in the absence or presence of 100 nM VD3 or with 100 nM 1,25VD, and myotube diameters were measured after further 24 h. (B) Representative images of the PLA-detected complexes of VDR and RXR (red) in C2C12 myotubes (green) treated for the indicated times with 100 nM VD3 or 100 nM 1,25VD. Nuclei were stained with DAPI. The white arrows indicate the nuclei of bone fide residual undifferentiated myoblasts. (C) Quantification of the PLA dots in myotubes (top) or myoblasts (bottom), in nuclei (left), cytoplasm (middle), and their ratio (right). (D) Myotube diameters of C2C12 treated with 20ng/mL IL6, with or without 100 nM VD3, or with 100 nM 1,25VD, in the presence/absence of 2.5 μM RXR inhibitor PA 452. (E) C2C12 myotubes were transfected with non-targeting or Pdia3 specific siRNAs, treated with 20ng/mL IL6 in the absence or presence of 100 nM VD3 and analyzed after further 24 h. Data are presented as the mean ± SEM. *P P P $P $$P

Figure 5. VDR, but not RXR nor 1,25D3-MARRS (Pdia3), mediates VD3 anti-atrophic activity. (A) C2C12 myotubes were transfected with non-targeting or Vdr specific siRNAs. 24 h after silencing, C2C12 myotubes were treated with 20ng/mL IL6 in the absence or presence of 100 nM VD3 or with 100 nM 1,25VD, and myotube diameters were measured after further 24 h. (B) Representative images of the PLA-detected complexes of VDR and RXR (red) in C2C12 myotubes (green) treated for the indicated times with 100 nM VD3 or 100 nM 1,25VD. Nuclei were stained with DAPI. The white arrows indicate the nuclei of bone fide residual undifferentiated myoblasts. (C) Quantification of the PLA dots in myotubes (top) or myoblasts (bottom), in nuclei (left), cytoplasm (middle), and their ratio (right). (D) Myotube diameters of C2C12 treated with 20ng/mL IL6, with or without 100 nM VD3, or with 100 nM 1,25VD, in the presence/absence of 2.5 μM RXR inhibitor PA 452. (E) C2C12 myotubes were transfected with non-targeting or Pdia3 specific siRNAs, treated with 20ng/mL IL6 in the absence or presence of 100 nM VD3 and analyzed after further 24 h. Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 vs. non-treated controls (NT); $P < 0.05; $$P < 0.01 vs. VD3-treated cells.