Fig. 8. IRF1 contributes to 2′3′-cGAMP/BV6/zVAD.fmk-induced cell death.

A mRNA expression levels of IRF1 were determined in the indicated cell lines subjected to 4 µg/ml 2′3′-cGAMP in the presence of 20 μM zVAD.fmk using qRT-PCR. Data are normalized to GAPDH expression and are presented as x-fold mRNA expression compared to control. Mean and SD of three independent experiments performed in triplicate are shown. ***P < 0.001. B Western blot analysis of IRF1 in indicated PC cell lines after 48 h of transfection with non-silencing RNA (siCtrl) or siRNA targeting IRF1 that were left treated with 5 µM BV6, 3 ng/ml IFNγ, and 20 µM zVAD.fmk for 15 h. β-Actin serves as loading control. Representative blots of at least two different independent experiments are shown. C AsPc-1 and BxPc-3 cells were transfected with control siRNA (siCtrl) or two independent siRNAs targeting IRF1 and were treated with 4 µg/ml 2′3′-cGAMP and 5 µM BV6 for 48 h in the presence of 20 μM zVAD.fmk. The amount of cell death was calculated by quantifying PI uptake determined with the ImageXpress Micro XLS system. Data are presented as percentage of PI-positive cells, and mean and SD of three independent experiments performed in triplicate are shown. **P < 0.01, ***P < 0.001. D Western blot analysis of nHT and IRF1 CRISPR/Cas9 KO in indicated PC cell lines that were treated with 5 µM BV6, 3 ng/ml IFNγ, and 20 µM zVAD.fmk for 15 h. β-Actin serves as loading control. Representative blots of at least two different independent experiments are shown. E AsPc-1 and BxPc-3 cells were subjected to nHT and IRF1 CRISPR/Cas9 knockdown, and treated with 4 µg/ml 2′3′-cGAMP and/or 5 µM BV6 for 48 h in the presence of 20 μM zVAD.fmk. The amount of cell death was calculated by quantifying PI uptake determined with the ImageXpress Micro XLS system. Data are presented as percentage of PI-positive cells and mean and SD of three independent experiments performed in triplicate are shown. **P < 0.01, ***P < 0.001.