Fig. 7

AAR pathway induced by halofuginone triggers mTOR degradation. a WRO cells were exposed or not to 100 nM halofuginone (HF) in the presence or in the absence of additional 2 mM proline (PRO). After 8 h, the cell homogenates were subjected to immunoblotting to assess total mTOR protein level and its phosphorylation status (at Ser 2448 and Ser 2481). The same set of samples was loaded in three different gels in order to avoid multiple stripping and re-probing of the same filter. Each filter was probed with anti β-Tubulin antibody as loading control. The pattern of protein expression shown was reproduced in three separate experiments. Densitometry of the protein bands was performed and mTOR/Tubulin ratios are included. b WRO cells adherent to coverslips and treated as in (a) were fixed, processed for mTOR (green) and LAMP-1 (red) immunostaining and imaged by confocal fluorescence microscopy. Nuclei were stained with DAPI. Scale bars: 10 μm. The images shown are representative of three separate experiments. c Bars indicate the average yellow fluorescence intensity density of immunofluorescences shown in (b). Data are from five different images for each condition. Error bars: standard deviation. Statistically significant differences between fluorescence intensity densities after to before HF, or after to before PRO in the presence of HF are shown (*, p ≤ 0.05)