Tomatidine

All animal care and treatments were done as approved by Animal Use and Care Committee of Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH). Adult Xenopus laevis were purchased from NASCO. Tadpoles were obtained by breeding wild type frogs, and were staged according to [64 ].
Stage 58 Xenopus laevis tadpoles of similar sizes were treated with cyclopamine (Enzo life sciences) (2.5 μM), tomatidine (Enzo life sciences) (2.5 μM), or vehicle (100% ethanol, final concentration in rearing water: 0.1%), respectively, at room temperature. The rearing water was changed every two days.

We obtained tomatidine from Enzo Life Sciences; rapamycin from Sigma; and [³H]tyrosine (40 Ci/mmol) from American Radiolabeled Chemicals. Anti-troponin (CT3), anti-myosin heavy chain (MyHC) I (BA-F8), anti-MyHC IIa (SC-71), anti-MyHC IIb (BF-F3), and anti-MyHC IIx (6H1) mouse monoclonal antibodies were obtained from the University of Iowa Developmental Studies Hybridoma Bank. Alexa Fluor 488-conjugated anti-mouse IgG₁, Alexa Fluor 350-conjugated anti-mouse IgG_2b and Alexa Fluor 555-conjugated anti-mouse IgM were obtained from Invitrogen. Anti-PGC-1α1 rabbit polyclonal antibody (ab54481) was obtained from Abcam. All other antibodies were obtained from Cell Signaling Technologies (anti-S6K (2308S), anti-phospho-S6K (Thr-389) (9234S), anti-Akt (4691S), anti-phospho-Akt (Ser-473) (4060S), anti-α-tubulin (2125S), and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (7074S)).