Fig. 1

Cell growth, viability, enzymatic activities and JNK phosphorylation in L6C5 myoblasts under serum starvation condition. a MTS viability assay. Cell growth values are expressed as fold increase in absorbance (OD) values after normalization according to values obtained at 6 h from the seeding, when cells usually become adherent to the plate and start growing (NGF 100 ng/ml: 2.2- to 5.21-fold increase vs Ctrl: 1.1- to 3.27-fold increase, p < 0.05). b Trypan blue exclusion assay. Analysis of viable, trypan blue negative (white) and nonviable, trypan blue positive (black) cells was performed after 48 h with or without NGF (10 and 100 ng/ml) and it is expressed as total number of counted cells (Viable cells: Ctrl, 8.8 ± 2.5 × 10³; NGF 10 ng/ml, 10.1 ± 3.6 × 10³; NGF 100 ng/ml, 9.2 ± 2.2 × 10³; p > 0.05; Nonviable cells: Ctrl: 1.3 ± 0.6 × 10³; NGF 10 ng/ml: 3.3 ± 1.9 × 10³; NGF 100 ng/ml: 2.1 ± 2.6 × 10³; p > 0.05). Three independent experiments were performed in triplicate. c ALT, HAD, LDH, CS and GAPDH enzyme activities performed in L6C5 myoblasts grown in serum-free medium and treated with NGF for 48 h. Data are shown as fold increase with respect to their control and expressed in percent values. One unit of enzymatic activity was defined as the amount of enzyme that forms 1 µmol of product per minute per mg of tested protein. The values have been collected from three independent experiments, each performed in triplicate. *p < 0.05. d Analysis of JNK activation on L6C5 myoblasts treated with or without NGF under serum starvation conditions. Immunoblot analysis of phosphorylated and total JNK in L6C5 cells at different times (15, 30 and 60 min) since NGF (20 ng/ml) supplementation. The histogram represents phospho JNK/total JNK ratio as mean ± SD of experiments repeated at least three times. *p < 0.05