FIGURE 10.

FZ induces ER stress response in human cancer cells. A, H460 cells were treated with FZ for 24 h, total RNA was isolated, and quantitative PCR was performed for analyzing relative gene expression for ER stress-specific genes. B, 293T cells were transfected with pSEAP-con plasmid, and on the following day fresh media containing 1% serum and specified concentrations of FZ, bortezomib, or MG132 were added. SEAP activities were determined as described under “Experimental Procedures” after incubation for 24 h. C, H460 cells were treated with 1 μm FZ, 1 μm bortezomib, or 10 μm MG132 for 24 h, following which RNA was isolated and reverse transcribed, and semi-quantitative PCR was performed using specific primers for the detection of two different splice variants of XBP1. D, Western blotting for BCL2, phospho-JNK (24 h) and p38 MAPK in H460 cells at different time points after FZ treatment. E, phase-contrast images of H460 cells after treatment with FZ alone or in the presence of cycloheximide (CHX). H460 cells were seeded at a density of 2 × 10⁴ cells/well in 24-well culture plates and then left untreated or treated with 5 μm FZ either alone or in the presence of 10 μg/ml cycloheximide for 24 h. F, cells were then trypsinized, and viable cells were counted by the trypan blue exclusion method. All experiments were done in triplicate. Error bars, S.D.