FIG 3.

Effects of IRE1α and XBP1 knockdown on IBV-induced apoptosis. (a) Effects of IRE1α and XBP1 knockdown on IBV-induced PARP cleavage. H1299 cells in duplicate were transfected with siIRE1α, siXBP1, or siEGFP. At 48 h posttransfection, the cells were infected with IBV (MOI, ∼2) or mock infected. One set of cells was harvested at the indicated time points and subjected to Western blot analysis using antibodies against IRE1α, XBP1, IBV N, and PARP. β-Tubulin was included as a loading control. The percentage of PARP cleavage [PARP Clv. (%)] was calculated as the intensity of cleaved PARP [PARP(Cl)] divided by the total intensities of full-length PARP [PARP(FL)] and PARP(Cl). In the second set of cells, total RNA was extracted and subjected to RT-PCR using primers specific for XBP1 and GAPDH. (b) Quantification of PARP cleavage in siRNA-transfected cells infected with IBV. The percentage of PARP cleavage in cells transfected with siIRE1α, siXBP1, or siEGFP and infected with IBV for 22 h was determined as for panel a. The bar chart shows the results from three independent experiments with standard deviations and P values. (c) Effects of IRE1α and XBP1 knockdown on IBV-induced caspase activation. The IBV-infected, 22-h p.i. protein samples and the siEGFP-transfected mock-infected protein sample from panel a were subjected to Western blot analysis using antibodies against IBV N, caspase 8, caspase 3, and caspase 9. β-Actin was included as a loading control. The percentage of caspase cleavage was calculated as the intensity of cleaved caspase divided by the total intensities of full-length and cleaved caspase. (d) The culture supernatants from IBV-infected samples in panel a were clarified by centrifugation and subjected to plaque assay analysis using a confluent monolayer of Vero cells. Virus titers are expressed as the logarithm of PFU per milliliter of supernatants. The experiment was repeated three times with similar results, and the result of one representative experiment is shown.