Figure 5

ATF3 negatively regulates cellular autophagy. (a) Neuro2a or MEF cells were treated with either a control or Atf3-specific siRNA for 48 h and then infected with JEV (MOI 5). Total proteins were harvested at 24 h pi as cell lysates. Western blotting of LC3 was performed to study the induction of cellular autophagy. Shown below is the ratio of LC3-II/GAPDH. (b) Neuro2a cells were treated with either a control or Atf3-specific siRNA for 48 h and then infected with JEV (MOI 5). Total RNA was isolated from the cells at 24 h pi and qRT PCR performed to determine the relative transcript levels for various autophagy-regulated genes. Mean values were used to create the heat map demonstrating the gene-expression profiles using the Gene-E software. (d) Neuro2a cells were treated with either a control or Atf3-specific siRNA for 48 h and then infected with JEV (MOI 5). Relative expression of various genes at different times pi was studied by qRT PCR, and the mean value is plotted. (e) MEF cells were treated with either a control or Atf3-specific siRNA for 48 h and then transfected with 1 μg/ml poly(IC). Total RNA was isolated from the cells at 24 h post-transfection, and qRT PCR performed to determine the relative transcript levels of various autophagy-regulated genes, and the mean values were used to create the heat map demonstrating the gene-expression profiles using the Gene-E software. (f) MEF cells were treated with either a control or Atf3-specific siRNA for 48 h and then infected with JEV (MOI 5) for 24 h. Relative expression of various autophagy-related genes was studied by qRT PCR, and the mean value is plotted. (g) MEF cells were treated with either a control or Atf3-specific siRNA for 48 h and transfected with 1 μg/ml poly(IC). Relative expression of various autophagy-related genes was studied 24 h later by qRT PCR and the mean value is plotted. Transcript level in control siRNA-treated cells is shown by the black bar while that in the Atf3 siRNA-treated cells is shown by the gray bar. The Gapdh levels were used for normalization. All the experiments were done in triplicates on three different occasions.The statistical significance of the difference between the data was established by Student’s t-test; *p < 0.05; **p < 0.01; ***p < 0.001.