Figure 2.

Reolysin induces ER stress. (a) Reovirus replication in Panc-1 cells. Cells were treated with 100 PFU/cell Reolysin for 48 h. Reovirus replication was detected by immunocytochemistry and electron microscopy. (b) Reolysin does not promote PERK or eif2α phosphorylation. Panc-1 cells were treated with 100 PFU/cell Reolysin for 24 and 48 h or with 5 μg/ml tunicamycin (24 h) as a positive control. Proteins were detected by immunoblotting. (c) Reolysin promotes ER swelling. Panc-1 cells were treated with 100 PFU/cell Reolysin for 48 h, and ER morphology was visualized by electron microscopy. Arrows denote endoplasmic reticulum. (d) Reolysin treatment increases intracellular calcium levels. Panc-1 cells were treated with the indicated amounts of Reolysin for 16 h, and intracellular calcium levels were detected by calcium green-1 staining and flow cytometry. Mean±S.D., n=3. *Represents a significant difference compared with controls. (e) qRT-PCR analysis of BiP, GADD34, CHOP, and XBP-1s expression in Panc-1 cells. Cells were treated with 100 PFU/cell Reolysin for 24 and 48 h and then harvested for analysis. Levels of mRNAs were standardized to the expression of GAPDH. Mean±S.D., n=3. *Indicates a significant difference from the control. P<0.05. (f) Immunoblotting analysis of CHOP, GADD34, BiP, PDI, ERp57, and calreticulin. Panc-1 cells were treated with 100 PFU/cell Reolysin for 24 or 48 h. ER-related protein expression was measured by immunoblotting