Chemical induction of ER stress significantly increased apoB-GFP-LC3 positive cells and the number of apoB-GFP-LC3 puncta (Fig. 4A; panels f and i; analysis of data shown in Fig. 4D,E; *P < 0.05 versus corresponding control). Endogenous LC3-II conversion (Fig. 4F; *P < 0.05 versus corresponding control) was significantly increased as compared to basal controls. The addition of 3-MA significantly decreased the number of apoB-GFP-LC3 positive cells and

the number of apoB-GFP-LC3 puncta (Fig. 4B, panels c, f, and i; and analysis of data shown in Fig. 4D,E; *P < 0.05 versus corresponding control), and endogenous LC3-II conversion (Fig. 4F; P < 0.05) under basal and ER stress conditions. By contrast, addition of the lysosomal protease inhibitor E64d, markedly increased the number of apoB-GFP-LC3 positive cells and the number of apoB-GFP-LC3 puncta (Fig. 4C, panels c, f, and Wnt beta-catenin pathway i; analysis of data shown in Fig. 4D,E; P < 0.05), as well as blocked endogenous LC3-II turnover (Fig. 4F; *P < 0.05 versus corresponding control). Taken together, these data further support the induction of apoB autophagy in a process that involves the formation of autophagosomes and accumulation in lysosomes for eventual proteolysis. To examine underlying mechanisms, mRNA levels of key molecules Pexidartinib in vitro in ER stress pathways were determined following 0, 2, 4, and 16 hours of

treatment with glucosamine (5 mM) or TM (5 μg/mL) in the presence or absence of PBA (1 mM) in McA-RH7777 cells. the mRNA levels of GRP78, PERK and ratio of spliced/unspliced form of Xbp-1 were significantly increased by 1.7-fold (*P < 0.05), 1.45-fold (*P < 0.05), and 4.23-fold (*P < 0.05), respectively, following glucosamine treatment (Fig. 5A,B). ATF6

mRNA level remained unchanged. PBA treatment markedly inhibited increases in mRNA levels of GRP78, PERK and ratio of spliced/unspliced form 上海皓元 of Xbp-1 (P < 0.05), suggesting that under our experimental conditions, PERK and IRE1, but not ATF6 signaling may be linked to apoB-autophagic degradation. Similar results were observed in cells treated with TM (Fig. 5C,D). To investigate the role that PERK activation may play in ER stress–induced apoB autophagic degradation, we cotransfected McA-RH7777 cells with GFP-LC3 cDNA and WT PERK cDNA, or kinase inactive (K618A) mutant (M) PERK cDNA, or control (mock), and examined the colocalization of apoB with GFP-LC3 following TM or glucosamine treatment. Under basal conditions (in the absence of ER stress–inducing agents), transfection with PERK WT cDNA led to a significantly increased number of apoB-GFP-LC3–positive cells and the number of apoB-GFP-LC3 puncta (Fig. 6A, panels c and f; analysis of data shown in Fig. 6D,E; *P < 0.05 versus mock), as well as elevated GFP-LC3-II conversion (Fig.