Journal: Nucleic Acids Research

Article Title: Loss of the DNA repair protein, polynucleotide kinase/phosphatase, activates the type 1 interferon response independent of ionizing radiation

doi: 10.1093/nar/gkae654

Figure Lengend Snippet: Involvement of the cGAS-STING signalling pathway in the T1IFN response following PNKP depletion. ( A ) Effect of RNAi-mediated double downregulation of PNKP and cGAS or ZBP1 on the T1IFN response in MCF7 cells. ( B ) Effect of cGAS inhibition by G140 on the T1IFN response in PNKP-depleted MCF cells. For the western blots shown in (A) and (B), whole cell extracts were used to determine protein expression. ( C ) Densitometric analysis of the of the western blot signal for pSTAT1/STAT1 ratio in (B) above. ( D ) Measurement of cGAS activity in PNKP-depleted MCF7 cells relative to the control cells. 2′3′-cGAMP was determined from cell lysates using an ELISA. All data are presented as standard error of the mean of n = 4 experiments (** P < 0.01, *** P < 0.001). Statistical significance was determined using unpaired Student's t -test.

Article Snippet: Inhibitors of cGAS (G140, Inh-g140), cyclosporin A (CSA, 12088), JAK1/2 inhibitor (Ruxolitinib, 11609), STING inhibitor (C-178, 25860) were purchased from Cayman Chemical (Ann Arbor, Michigan, USA).

Techniques: Inhibition, Western Blot, Expressing, Activity Assay, Control, Enzyme-linked Immunosorbent Assay

Journal: Nucleic Acids Research

Article Title: Loss of the DNA repair protein, polynucleotide kinase/phosphatase, activates the type 1 interferon response independent of ionizing radiation

doi: 10.1093/nar/gkae654

Figure Lengend Snippet: Schematic diagram illustrating the underlying mechanism of how loss of PNKP activates the type 1 interferon response. Left panel: in the presence of PNKP, ROS-induced DNA damage fails to activate the T1IFN response as the strand break termini are constantly repaired by PNKP. However, in the absence of PNKP, the strand breaks induced by ROS are persistent, leading to the generation of smaller DNA fragments that are bound by ZBP1 and/or cGAS. Middle panel: activated cGAS synthesizes the second messenger molecule 2′3′-cGAMP, which binds to and activates STING leading to the downstream phosphorylation and activation of TBK1, IRF3 and subsequent synthesis and secretion of type 1 interferons such as IFNβ. Likewise, ZBP1, bound by oxidized DNA, is activated, and in complex with cGAS augments the activity of cGAS and STING. Right panel: the secreted IFNβ binds to its cognate receptor thereby promoting downstream phosphorylation events involving sequential JAK/TYK1 activation, STAT1/STAT2 activation and complex formation with IRF9. This ISGF3 complex translocates to the nucleus where it turns on interferon-stimulated genes. (Figure created with BioRender.com).

Article Snippet: Inhibitors of cGAS (G140, Inh-g140), cyclosporin A (CSA, 12088), JAK1/2 inhibitor (Ruxolitinib, 11609), STING inhibitor (C-178, 25860) were purchased from Cayman Chemical (Ann Arbor, Michigan, USA).

Techniques: Phospho-proteomics, Activation Assay, Activity Assay