Melatonin, a novel selective ATF-6 inhibitor, induces human hepatoma cell apoptosis through COX-2 downregulation

Figure 4 Relationship between activating transcription factor 6 and cyclooxygenase-2 under endoplasmic reticulum stress. The mRNA of the unfolded protein response (UPR) pathways was interfered by ATF-6, IRE-1 and PERK siRNA after pretreatment by tunicamycin (TM) for 8 h. A: Equal protein amounts of cell lysates were subjected to western blot assay using anti-COX-2. β-actin in the same HepG2 cell extract was used as an internal reference. B: Optical density reading values of specific proteins are represented as fold-differences relative to the loading control protein, β-actin. ^aP < 0.01 vs the negative control. RNA was harvested and gene expression examined by qRT-PCR in the same condition above. C: The qRT-PCR fold-changes were normalized using the expression of housekeeping gene (GAPDH) and vs those obtained from untreated HepG2 cells. ^aP < 0.01 vs the negative control; ^cP < 0.01 vs the untreated HepG2 cells. ATF-6: Activating transcription factor 6; NC: Negative control; COX-2: Cyclooxygenase-2; IRE: Inositol-requiring enzyme; PERK: Protein kinase RNA-like endoplasmic reticulum kinase.