Figure 5
Upregulation of UPR proteins activate ER stress response, generation of reactive oxygen species and JNK activation, which in turn regulate autophagy. (A) PA-1 and OVCAR-3 cells were treated with ORM IC50 dose for indicated time-points and immunoblotted for UPR sensor proteins PERK, ATF6 and IRE1 as well as ER chaperone GRP78/BiP, downstream transcription factor CHOP/GADD153 and eukaryotic translation initiation factor eIF2α, apart from phosphorylated JNK (p-JNK) levels. (B) densitometric analyses of data represented in (A). (C) PA-1 and OVCAR-3 cells were grown in confocal glass-bottom dishes, treated with IC50 dose of ORM for 24 h followed by staining with CM-H2DCFDA, a live-cell ROS marker, and imaged in confocal microscope. (D) Quantification of fluorescence intensity of each cell from data such as (B) as described in Methods. (E) PA-1 cells were pre-treated with NAC (5 mM, 2 h) or Tiron (5 μM, 1 h), treated with ORM (24 h) and LC3 conversion was analysed by western blotting. (F) PA-1 and OVCAR-3 cells were pre-treated or not with 4-PBA (5 mM; 2 h) or 3-MA (2.5 mM; 2 h) before treating with IC50 dose of ORM and immunoblotting for GRP78 and PERK for quantification of ER stress and LC3 for quantification of autophagy. (G) PA-1 cells were pre-treated or not with small-molecule JNK inhibitor SP600126 (SP6; 10 μM,1 h) before IC50 ORM treatment and immunoblotted for LC3 as a marker of autophagy. (E) Scale bars = 10 μm.
