Figure 6

IFN-γ induces autophagy in BMECs via a mechanism dependent on GCN2. (a) Amino acids are metabolized during IFN-γ treatment. BMECs were incubated with 10 ng/ml IFN-γ for 6, 12 or 48 h or were left untreated. Sixteen types of free amino acids from the BMECs were detected by an amino acid analyser. The data represent the mean±S.E.M. of three independent experiments. Error bars are±S.E.M. One-way ANOVA; *P<0.05; **P<0.01. (b, c) BMECs were treated with different concentrations of IFN-γ (5, 10 or 20 ng/ml) for different times (6, 12 or 24 h). At the end of the treatment, the expression of p-GCN2, total GCN2 and GAPDH was analysed using western blot analysis with specific antibodies as described in the Materials and Methods section. The data represent the mean±S.E.M. of three independent experiments. Error bars are±S.E.M. One-way ANOVA; *P<0.05; **P<0.01. (d) BMECs were treated with IFN-γ (10 ng/ml). After 24 h, the cells were stimulated with arginine (13.6 mol/l) for 6 h. At the end of the treatment, the expression levels of GCN2, p-GCN2, MAP1LC3 and GAPDH were analysed using western blot analysis with specific antibodies as described in the Materials and Methods section. The data represent the mean±S.E.M. of three independent experiments. Error bars are ±S.E.M. One-way ANOVA; *P<0.05; **P<0.01. (e) Inhibition of GCN2 expression by adenovirus-delivered shRNA of GCN2. BMECs were infected with vAd-GFP-GCN2 or vAd-GFP-NT (NT represents the control shRNA). Twenty-four hours post-infection, total GCN2, p-GCN2, MAP1LC3 and GAPDH protein levels were analysed using western blot analysis with specific antibodies as described in the Materials and Methods section. The data represent the mean±S.E.M. of three independent experiments. Error bars are ±S.E.M. One-way ANOVA; *P<0.05; **P<0.01.