Figure 3

IFN-γ increased rates of autophagy in primary BMECs in vitro. (a) The expression of IFNGRs in BMECs. BMECs were treated with IFN-γ for 6, 24 or 48 h. At the end of the treatment period, the levels of IFNGR1 and IFNGR2 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. (b) BMECs were treated with IFN-γ for 3, 6, 12 or 24 h. At the end of the treatment, levels of MAP1LC3, ATG12-ATG5, SQSTM1/p62 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. (c) BMECs were treated with IFN-γ at 2.5, 5, 10 or 20 ng/ml. At the end of the treatment, levels of MAP1LC3, ATG12-ATG5, SQSTM1/p62 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. (d) Direct effect of IFN-γ on induction of autophagy. BMECs were cultured for 24 h in supernatants from either mock-treated BMECs or cells treated with 10 ng/ml of IFN-γ for 24 h in the presence or absence of anti-IFNGR1, IFNGR2 or IFNGR1/2. At the end of the treatment, levels of MAP1LC3, ATG12-ATG5, SQSTM1/p62 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.