Figure 6.

Curcumin treatment attenuated XBP1 splicing elicited by altered trafficking of the R98C mutant in Cos-7 cells. (A) Green fluorescence in the nucleus indicates the activation of the IRE1 pathway of the unfolded protein response in a cell culture reporter system where alternative splicing of XBP1 pulls GFP into frame. Representative epifluorescent images demonstrate that XBP1 splicing occurred more frequently in Cos-7 cells transfected with R98C than in those transfected with wild-type P0 and that curcumin treatment reduced the number of GFP-positive nuclei. Scale bar = 100 μm. (B) Confocal images illustrate that although wild-type P0 reached the cell surface, the R98C mutant was retained in the endoplasmic reticulum as well as elicited XBP1 splicing. At 2 μM curcumin treatment altered trafficking of P0R98C protein remained, but the number of GFP-positive nuclei decreased; whereas at 10€ μM curcumin treatment, the number of GFP-positive nuclei decreased but some mutant protein appeared to reach the cell surface, as every cell process and protrusion is labelled with haemagglutinin staining (see arrows). Scale bar = 10 μm. (C) Quantification of XBP1 splicing in vitro. The percentage of GFP-positive nuclei was significantly higher (P = 0.05) in Cos-7 cells transfected with the R98C mutant as in those transfected with the wild-type construct. In the end, 2 and 10 μM curcumin treatment reduced XBP1 splicing to the same extent (for both P = 0.05).