Figure 6.

Mutation of the LIR motif abolishes calreticulin-mediated augmentation of autophagy and reduction of ER stress. A and B, expression of LC3-II was not promoted by calreticulin ΔLIR mutation. HeLa cells were transfected with myc-CRT or the variant ΔLIR constructs, treated with tunicamycin (2 μg/ml, A) or thapsigargin (300 nm, B) for 16 h and without or with chloroquine (50 μm) for 10 h as indicated, and then lysed for immunoblotting. C, drug-induced ER stress was not attenuated by overexpression of calreticulin ΔLIR mutation. HeLa cells were transfected with gradient amounts of the indicated constructs, treated with vehicle (DMSO) or tunicamycin (2 μg/ml) for 16 h, and then lysed for immunoblotting. D, representative images of immunofluorescence (scale bars = 10 μm). HeLa cells were transfected with myc-CRT ΔLIR and GFP-LC3 constructs to detect autophagic flux. myc-CRT ΔLIR was labeled with red fluorescence. Cells were treated with vehicle (DMSO) or chloroquine (50 μm) for 10 h as indicated. Then immunofluorescence was performed, and quantification of the number of LC3 puncta is presented. Data are presented as the mean ± S.E.; n = 5. *, p < 0.05 versus vehicle.