FIGURE 2.

iPLA₂γ amplifies tunicamycin-induced ATF6 luciferase-reporter activity. A, rat GECs were transiently co-transfected with full-length (M1) GFP-iPLA₂γ, N-terminal truncated (M4) GFP-iPLA₂γ, or vector (control), plus ATF6 firefly luciferase-reporter and Renilla luciferase. After 24 h, cells were untreated (Untr) or incubated with tunicamycin (Tm, 0.1 μg/ml, 18 h). Tunicamycin stimulated ATF6 reporter activity (expressed in relative luminescence units (RLU)), and iPLA₂γ amplified the effect of Tm. *, p < 0.001 Tm versus untreated (Vector); **, p < 0.001 iPLA₂γ versus vector (Tm) and versus M4 iPLA₂γ (Tm); +, p < 0.01 Tm versus untreated (M4 iPLA₂γ), three experiments performed in triplicate. B, representative anti-GFP antibody immunoblots showing expression of full-length (M1) WT and N-terminal truncated (M4) GFP-iPLA₂γ (V, vector transfection). C, rat GECs were transiently co-transfected as in A. After 24 h, cells were untreated (Untr), incubated with tunicamycin (Tm, 0.1 μg/ml, 18 h), or preincubated with R-BEL (5 μm, 30 min) or indomethacin (10 μm, 30 min) before treatment with Tm. Tunicamycin stimulated ATF6-reporter activity, and iPLA₂γ amplified the effect of Tm. The amplified ATF6 activity was inhibited by R-BEL but not by indomethacin. *, p < 0.001 Tm versus untreated (Vector); **, p < 0.01 Tm + indomethacin (Indo) versus untreated (Vector); ***, p < 0.05 R-BEL + Tm versus Tm (Vector); +, p < 0.05 iPLA₂γ versus vector (Tm); ++, p < 0.001 R-BEL + Tm versus Tm (iPLA₂γ), five experiments performed in triplicate. D, GECs were co-transfected as in A. Cells were untreated or incubated with thapsigargin (Tg, 0.5 μm, 18 h) or DTT (0.5 μm, 18 h). Thapsigargin and DTT stimulated ATF6 reporter activity, and iPLA₂γ amplified the effect of thapsigargin and DTT. *, p < 0.001 thapsigargin versus untreated (Vector); **, p < 0.05 DTT versus untreated (Vector); +, p < 0.05 iPLA₂γ versus vector (Tg). ++, p < 0.05 iPLA₂γ versus vector (DTT), four experiments performed in triplicate.