Figure 5

TGF-beta1 effect on autophagy in L6 and C2C12 cells. (a) Differentiating L6 cells were treated with 5 ng/ml TGF-beta1 as described under Materials and methods. After 6 days in culture, the cells were stained with acridine orange and AVO accumulation was determined by flow cytometric analysis. Calculated percentages represent the red-positive cells in the upper quadrants (R10 and R11). FITC Lin, green color intensity; PE Texas Red Area, red color intensity. Those shown are representative of four independent experiments. Alternatively (b and d), L6 and C2C12 cells were lysed as described under Materials and Methods and western blotted using PED/PEA-15, Beclin1, LC3 I, LC3 II, phospho-Tyr307-PP2A, phospho-Ser256-FoxO1 or FoxO1 antibodies. Blots were revealed by ECL and autoradiographs, using beta-actin as loading control. The autoradiographs shown are representative of three independent experiments. (c) L6 and C2C12 cells were plated and exposed for 72 h to 5 ng/ml TGF-beta1 in the absence or the presence of 1 mM 3-methyladenine (3MA). Upon 6 days of differentiation, total RNA was isolated and the levels of MyoD1, Myogenin and Glut4 mRNA were quantitated by real-time RT-PCR analysis, with each reaction performed in triplicate. Bars represent the mean ±S.D. of four independent experiments. Asterisks denote statistically significant differences (^*P<0.05; ^**P<0.01; ^***P<0.001)