Figure 4. MA-mediated ER stress involved activation of PERK pathway.

SVGA cells were seeded in 12-well plates at 2.5 × 10⁵ cells/well and treated with 500 μM MA. For all the experiments involving siRNA transfection, the cells were seeded in 6-well plate and transfected with various siRNA as described in materials and methods prior to treatment with MA. A. The levels of phosphorylated PERK, phosphorylated eIF2α and ATF4 were increased as a result of MA treatment. B-C. Knockdown of PERK using siRNA reduced the levels of MA-mediated CHOP mRNA (B) and protein (C). D-E. Similarly, knockdown of ATF4 also reduced the CHOP mRNA (D) and protein (E). F. The levels of ATF4 were measured after knockdown of PERK to demonstrate a link between PERK activation and increase in ATF4 (F). The RNA and protein expressions in all the experiments were normalized with HPRT and GAPDH as housekeeping genes, respectively. The results shown in bar graphs were obtained from at least 3 independent experiments with each treatment performed in triplicates. The bar graphs shown in the figure are represented in mean ± S.E., while the western blots are representative images. The numbers above the blots represent mean intensity of the respective bands. Statistical significance was calculated using one-way ANOVA with multiple comparisons and the values were considered significant if p-value ≤ 0.01 (**).