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. 2023 Jul 20;15(14):3689. doi: 10.3390/cancers15143689

Figure 4.

Figure 4

Nuclear factor κB (NF-κB) is involved in the increased type I IFN signaling observed in EO771 Parp7KO cells. (A) Inhibition of TANK-binding kinase 1 (TBK1) with MRT67307 significantly decreases Ifnb1 mRNA levels in EO771 Parp7KO cells but not in WT cells. Inhibition of both TBK1 and the IκB kinases (IKKs) with BX795 blocks Ifnb1 mRNA expression in all cases. Cells were treated with either 1 µM of MRT67307 or 100 nM of BX795 for 24 h, followed by 10 µg/mL of DMXAA for 2 h. (B) Treatment with DMXAA and/or RBN-2397 increases protein levels of both p50 and RelA in EO771 WT cells while levels are higher in Parp7KO cells in both untreated and treated cells. Cells were treated with 10 µg/mL of DMXAA (+/− 100 nM of RBN-2397) for 24 h and proteins were visualized by Western blotting. (C) Levels of expressed NF-κB subunits are higher in response to PARP7 inhibition, and levels of RelA are significantly higher in the Parp7KO cells. Protein levels of p50 and RelA in untreated samples were quantified by normalizing to the loading control. (D) Expression levels of Rela mRNA are not affected by DMXAA or PARP7. (E) Knockdown of either TBK1, RelA, or both, in EO771 WT and Parp7KO cells. After 48 h of transfection with 25 nM of siRNA targeting Tbk1, Rela, or both, protein levels were visualized by Western blotting. (F) Knockdown of TBK1 and RelA results in significantly decreased Ifnb1 mRNA levels in Parp7KO cells but not in WT cells. After 48 h of transfection with 25 nM of siRNA for Tbk1, Rela, or both, cells were treated with 10 µg/mL of DMXAA for 2 h. (G) p50 and RelA interact with WT and catalytically inactive PARP7, and RelA is MARylated by WT PARP7. Cos-1 cells were transfected with FLAG-tagged p50 or RelA together with either GFP-tagged WT PARP7 or the catalytically inactive H532A mutant. The FLAG-tagged p50 or RelA was immunoprecipitated, and the interaction and modification was examined by Western blotting. * Denotes statistical significance (p < 0.05) compared to DMSO; # denotes statistical difference due to the loss of PARP7; “a” denotes statistical difference compared to no inhibitor (A) or to the nontargeting siRNA (F). Original western blots have been presented in File S1.