Figure 7. ATF3 negatively regulates select autophagy genes to influence autophagic flux.

A549 cells WT and ATF3 KO cells were mock-infected or infected with ZIKV PRVABC (moi=1 and 10 PFU/cell) and the expression of select autophagy genes examined 24-hours post infection. (A-D) RT-qPCR analyses of autophagy related genes ATG5, ATG12, ATG101 and ULK2 normalized to β-actin mRNA. N=3, Error bars show ± SD. Statistical significance was determined by Student T-test. *p<0.01, **p<0.001, ***p<0.0005. (E-F) A549 WT and ATF3 deficient cells were exposed to starvation media for 1-, 2- and 4 hours. Autophagy markers LC3B and p62/SQSTM1 were examined by western blotting with GAPDH as the loading control. (G-H) A549 WT and ATF3 KO cells were mock-infected or infected with ZIKV PRVABC (moi=1 and 10 PFU/cell) and autophagy-associated proteins LC3B-I, LC3B-II and p62/ SQSTM1 were analyzed by western blot at 24-hours post infection. Immunoblots shown are representatives from 3 independent experiments. (I) p62/ SQSTM1 gene expression relative to β-actin was examined by RT-qPCR. In (G) cells were infected with ZIKV at moi=1 and 10 PFU/cell, while in (H-I) cells were infected with ZIKV at moi=10 PFU/cell. N=3, Error bars show ± SD. Statistical significance was determined by Student T-test. *p<0.01, **p<0.001.