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. 2018 Nov 12;8:16678. doi: 10.1038/s41598-018-34891-w

Figure 1.

Figure 1

Silencing of COX-2 affects IRE1α activity. (A) HEK293 cells were transfected with the IRE1α splicing reporter plasmid and treated for 24 hours with 20 µM cyclosporine A (+CsA). ****p-value < 0.0001 (n = 35). (B) Cells were transfected with the IRE1α splicing reporter plasmid in combination with siRNA for COX-2 (COX-2 siRNA) or control scrambled siRNA (scrambled siRNA) followed by treatment for 24 hours with 20 µM cyclosporine A (+CsA). ****p-value < 0.0001 (n = 21). (C) Q-PCR quantitative analysis of spliced endogenous XBP1 in HEK293 cells transfected with siRNA for COX-2 (COX-2 siRNA) or control scrambled siRNA (scrambled siRNA) followed by treatment with 20 µM cyclosporine A (+CsA). ****p-value < 0.0001 (n = 34). (D) HEK293 cells were transfected with siRNA for COX-2 (COX-2 siRNA) or control scrambled siRNA (scrambled siRNA) and with the XBP1 splicing reporter vector. Cells were treated for 24 hours with 20 µM cyclosporine A (+CsA), thapsigargin (0.5 µM) (Thap), tunicamycin (5 µg/ml) (Tun) or DTT (1 mM). ****p-value < 0.0001, **p-value = 0.0014 (n = 16); NS, not significant. (E) Immunoblot analysis of HEK293 cells treated for 24 hours with 20 µM cyclosporine A (+CsA). Blots were probed with anti-COX-2 and anti-GAPDH antibodies. (F) HEK293 cells were incubated with 20 µM cyclosporine A (+CsA) followed by analysis of COX-2 peroxidase activity. ****p-value < 0.0001 (n = 20). (G) Peroxidase activity of purified COX-2 protein was monitored in the absence (no CsA) and presence of 20 µM cyclosporine A (+CsA). ****p-value < 0.0001 (n = 6). The images of (E) shown are cropped. The full-length blots are shown in Suppl. Fig. S10.