Figure 8.

Effect of an ER stress inhibitor on expression of autophagy-related genes and proteins. Cells were treated with OXY (10 μg/mL) with or without the ER stress inhibitor 4-PBA. Expression levels of IRE1α, XBP1, PERK, ATF4, Beclin-1, Atg5, Atg7, and LC3B were determined by qPCR (A). Expression levels of Beclin-1, Atg7, Atg5, and LC3B proteins were measured by Western blotting (B) and quantified (C). From each lysate, equal amounts of protein were loaded on a separate gel, blotted for actin and this signal was used for determining the ratio of the protein of interest/actin as displayed in the figure. The cropped blots are representative of three independent experiments. The full-length blots are shown in Supplementary Figure S6. Autophagic vesicle formation was detected using the CYTO-ID autophagy green dye and observed by fluorescence microscopy; white arrows indicate autophagosomes (magnification 600×) (D), which were quantified (E). Each value indicates the mean ± SD of three independent experiments performed in triplicate.