Figure 6.

Amiloride (Ami) did not induce CCAAT/enhancer-binding protein homologous protein (CHOP) expression. (A) Cells were treated with tunicamycin (Tm: 0.01 µg·mL⁻¹) or Ami (200 µM) for the periods indicated. Although Ami increased eIF2α phosphorylation, it did not induce CHOP expression. Western blotting was performed using anti-phospho-eIF2α (42 kDa), anti-eIF2α (40 kDa), anti-CHOP (29 kDa) and anti-GAPDH (37 kDa) antibodies. (B) Cells were treated with Ami (20–500 µM) for 1 h and then stimulated with Tm (0.01 µg·mL⁻¹) or dithiothreitol (DTT: 3 mM) for 6 h. Reverse transcription-polymerase chain reaction was performed using specific primers for CHOP and GAPDH. (C) Cells were treated with Ami (20–500 µM) for 1 h and then stimulated with Tm (0.01 µg·mL⁻¹) or DTT (3 mM) for 18 h. Western blotting was performed using anti-CHOP (29 kDa) and anti-GAPDH (37 kDa) antibodies. (D–G) Densitometric analysis of CHOP mRNA and protein using image analysing software. Ami dose-dependently inhibited ER stress-induced CHOP expression. *P < 0.05, **P < 0.01, ***P < 0.001 versus Tm or DTT (n= 6 for RT-PCR, n= 5 for Western blotting). Cont, control.