Figure 2.

GRP78 regulates CSE-induced necroptosis.

Notes: (A) HBE cells were transfected with control or GRP78-siRNA for 24 h and then were treated with 4% CSE for an additional 24 h. Cell death was determined by CCK8 assay. (B) HBE cells were cultured with NEC-1 (50 μM) or vehicle (DMSO) together with 4% CSE for 24 h. Cell death was determined by CCK8 assay. (C) HBE cells were exposed to 4% CSE at indicated times. Expressions of RIP1, RIP3, and p-MLKL were measured by Western blotting. MTECs were treated with 4% CSE at indicated times, and the protein levels of RIP1 and RIP3 were assessed by Western blotting (D). Wild-type mice were exposed to room air or CS, and protein levels of RIP1 and RIP3 from mouse lung homogenate samples were measured by Western blotting analysis (E). HBE cells were pretreated with control or GRP78-siRNA for 24 h. After that, cells were incubated with 4% CSE for an additional 12 h, and cell lysates were then subjected to Western blotting for RIP1, RIP3, p-MLKL, and GRP78 (F). Data (A–F) were mean ± SEM of three independent experiments. Western blots (C–F) were representative of three independent experiments. *P<0.05 and **P<0.01 (Student’s t-test).

Abbreviations: CS, cigarette smoke; CSE, cigarette smoke extract; GRP78, glucose-regulated protein 78; HBE, human bronchial epithelial; MTECs, mouse tracheal epithelial cells; CCK8, cell counting kit 8; NEC-1, necrostatin-1; DMSO, dimethyl sulfoxide; RIP, receptor-interacting protein; SEM, standard error of the mean.