Figure 1

Ethanol extract of the P. calophylla stem inhibits activation of the IRE1-XBP1 pathway. (a) The scheme of the XBP1 splicing system (XBP1us-luc2 and XBP1s-GFP) in the reporter construct is shown. (b) HEK293 cells, stably expressing both XBP1us-luc2 and XBP1s-GFP, were pretreated with the ethanol extract of the P. calophylla stem (P. calophylla EtOH ex.) (100 µg/ml) for 1 h, and were then incubated with tunicamycin (TM) (0.5 µg/ml) or thapsigargin (TG) (0.5 µM). DMSO was added as vehicle. After 6 h, luciferase activities and fluorescence intensities in cell lysates were measured. The luciferase activity was normalized by the fluorescence intensity. Values are shown as mean fold activity ± standard deviations (S.D.) (n = 3 biological replicates). Statistical analysis was conducted between two groups with or without P. calophylla EtOH ex. for each TM or TG treatment assessed by two-tailed Student’s t-test, and the significant differences are indicated as *p < 0.05. (c) HEK293 cells were pretreated with the P. calophylla EtOH ex. in a dose-dependent manner (10, 30, 100 µg/ml) for 1 h, and were then incubated with TM (0.5 µg/ml) for 6 h. The expression of each gene was assessed by RT-PCR. Unspliced or spliced XBP1 mRNA are indicated. GAPDH was used as the loading control. Uncropped images of gels are shown in Supplementary Information, Fig. S7.