The Role of Taurine on Skeletal Muscle Cell Differentiation - Advances in Experimental Medicine and Biology - page 302

the cells are cultured in the taurine-deficient condition. The present study examined
the effect of taurine treatment on the differentiation of mouse myoblast to myotube.

29.2 Methods

29.2.1 Culture and Differentiation of Myoblast Cells

Mouse differentiable myoblast (C2C12) was purchased from ATCC (Manassas, VA).
C2C12 cells were cultured with growth medium (GM; DMEM supplemented with
10% fetal bovine serum) until confluent, and thereafter, the cells were switched to
differentiation medium (DM; DMEM supplemented with 2% horse serum) with or
without ~20 mM taurine for up to a week. Furthermore, the cells were exposed to
~50 mM taurine transport agonist, b-alanine; 5 mM calcineurin inhibitor, FK-506
(Cayman chemical, MI, USA); or 10 m M Ca 2+ chelator, nifedipine (Sigma, MO,
USA) in DM with or without 20 mM taurine for 5-6 days.
In C2C12 myoblast, taut mRNA was also silenced using siRNA (HP GenomeWide
siRNA duplexes; QIAGEN, Hilden, Germany) by electroporation method (Amaxa™
Nucleofector™ Technology, Lonza, Cologne, Germany). Harvested undifferenti-
ated C2C12 myoblast (1 × 10 6 cells) was transfected with the siRNA primers of taut
or control and pmaxGFP ® vector (Lonza), and the cells were grown up to confluent
with GM. Thereafter, the cells were differentiated in DM with or without taurine.

29.2.2 Quanti fi cations of Cell Size

and Nuclei Number in Myotube

The differentiated C2C12 myotube cultured with and without taurine was fixed with
methanol and 4% paraformaldehyde, and then, myosin heavy-chain (MHC) protein
as a marker of differentiation in the skeletal muscle was immunohistochemically
detected using monoclonal anti-skeletal MHC-fast antibody (Sigma) as the primary
antibody and goat anti-mouse IgG antibody conjugated with FITC (Santa Cruz
Biotechnology, CA, USA) as the secondary antibody, and the nucleus was labeled
with DAPI (KPL, MD, USA). FITC and DAPI were observed using a fluorescence
microscopy, and maximal short diameter and apsis length of FITC-positive in dif-
ferentiated myotube and the number of DAPI-positive nucleus in the FITC-positive
myotube were measured, and the number of nucleus per myotube and fusion index
as ratio of the number of nucleus to the apsis length of myotube were calculated.
In the C2C12 myotube transfected with taut siRNA, the transfected GFP that
is a marker of silenced taut gene was detected using fluorescence microscopy.
The efficiency of silencing of taut gene was approximately 70% evaluated as TAUT
protein expression by Western blot.