Figure 2.
Characterization of the response of SEAP to ER stress. (A) Rapid reduction in SEAP activity by ER stress. LL/SEAP cells were pretreated with thapsigargin (1 µM) or A23187 (2 µM) for 30 min. After changing the medium, the cells were further incubated in the presence of the drugs for up to 60 min. Culture media were collected every 15 min and subjected to SEAP assay. (B) Responses to known ER stress inducers. LL/SEAP cells were treated with tunicamycin (Tun; 10 µg/ml), thapsigargin (Thap; 1 µM), A23187 (2 µM), dithiothreitol (DTT; 1 mM), cobalt chloride (Cobalt; 1 mM) or MG132 (50 µM) for 6 h and subjected to northern blot analysis of grps (left) and SEAP assay (right). (C) Lack of responses to ER stress-unrelated stimuli. LL/SEAP cells were treated with interleukin 1β (IL-1β; 20 ng/ml), tumor necrosis factor-α (TNF-α; 250 U/ml), transforming growth factor-β (TGF-β; 10 ng/ml) or 12-o-tetradecanoylphorbol-13-acetate (TPA; 50 ng/ml) for 6 h and subjected to analyses. Tunicamycin (10 µg/ml) and thapsigargin (1 µM) were used as positive controls. (D) Secretion of SEAP by Hepa1/SEAP and SM/SEAP cells. The murine hepatoma cell line Hepa-1c1c7 and the rat mesangial cell line SM43 were stably transfected with a SEAP gene under the control of the SV40 promoter, and Hepa1/SEAP and SM/SEAP cells were established. Activity of SEAP in culture media was evaluated by chemiluminescent assay. (E) Responses to ER stress of LL/SEAP and Hepa1/SEAP cells. LL/SEAP, Hepa1/SEAP and SM/SEAP cells were treated with (+) or without (−) thapsigargin (1 µM) for 6 h, and culture media were subjected to SEAP assay. Assays were performed in quadruplicate, and data are expressed as means ± SE. Asterisks indicate statistically significant differences (P < 0.05).