Fig. 5. OPTN knockdown causes activation of ATF4 and other members of the ER stress-signaling pathway, chaperone-mediated autophagy and apoptosis.

a Heat map of genes in the ATF4 network (IPA software) for Miapaca and Suit2-007 cells with the respective fold changes in expression. b WB analysis of the different antibodies in the ER stress-signaling pathway including pEIF2α, ATF6, pPERK and ATF4 as well as cleaved PARP. c Miapaca cells stained with Annexin V-FITC show increased numbers of apoptotic cells in the OPTN KD sample as compared with nonspecific control and as detected from the percentage of gated cells in the lower right quadrant. d Staining of Miapaca cells with Hoechst 33248 stain 48 h following transfection with siRNA. OPTN samples show nuclear abnormalities in terms of shrinkage and fragmentation. e Analysis of the expression of HSP90 and LAMP2 as regulators of chaperone-mediated autophagy (CMA) by immunoblotting 48 h post transfection with siRNA. Analysis of the band density was done by ImageJ software and corrected to β-actin as loading control and to negative siRNA as the main control