Fig. 5

Excess of proline rescues the induction of AAR and of autophagy by halofuginone. a WRO cells were treated with 100 nM halofuginone (HF) in standard medium supplemented or not with 2 mM proline (PRO) as indicated. A representative immunoblotting of P-eIF2α versus total eIF2α (marker of AAR) and of LC3B (marker of autophagy) is shown. Densitometry analysis of the protein bands was performed and the LC3B-II/ I band density ratios are shown. A similar pattern of protein expression was observed in two other separate experiments. b WRO cells were plated on coverslips and treated as in (a). After 8 h the cells were fixed, processed for LC3 (green) and LAMP-1 (red) immunostaining and imaged by fluorescence microscopy. Nuclei were stained with DAPI. Scale bars: 10 μm. 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)