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: Loss of PNKP promotes IFNβ1 secretion that mediates downstream STAT1 activation. ( A ) Immunoblots showing activation of the T1IFN response as determined by elevated IRF3 and STAT1 phosphorylation following depletion of PNKP in MCF7 cells (first 4 lanes). Secreted interferons/cytokines present in conditioned media (CM) from PNKP-depleted MCF7 cells activate STAT1 independent of radiation (last four lanes). Naïve MCF7 cells were incubated with CM from the corresponding first four lanes) for 24 h prior to cell harvesting. ( B ) CM from PNKP-depleted MCF7 cells stimulate the T1IFN response in other naïve cancer cells. Cells were allowed to attach before 24-hour incubation with CM from PNKP-depleted MCF7 cells. ( C ) ELISA assay to determine levels of secreted IFNβ1 in media supernatants in PNKP-depleted MCF7, T47D and PANC-1 cells relative to the siControl cells 90 h post transfection with siRNA (replicates: n = 5 for MCF7, and n = 4 for both T47D and PANC-1). Data are presented as standard error of the mean of n number of experiments (* P < 0.05, ** P < 0.01 and *** P < 0.001). Statistical significance was determined using unpaired Student's t -test. ( D ) Western blot analysis showing result of neutralizing antibodies (NA) against IFNβ and IFNAR2 in PNKP-depleted MCF7 cells. ( E ) Western blot analysis showing result of neutralizing antibodies against IFNβ, IFNα and IFNAR2 in PNKP-depleted MCF7 cells; time of incubation = 24 h. For the western blots data shown in (A–E) whole cell extracts were used to determine protein expression. ( F ) Subcellular fractionation of MCF7 cells reveals the localization of proteins that mediate T1IFN response following downregulation of PNKP. Fractionation purity was assessed using lamin A/C and nucleolin as nuclear markers, and tubulin and GAPDH as cytoplasmic markers. Whole cell, cytosolic and nuclear extracts were used to determine protein expression.

Article Snippet: Primary antibodies to the following proteins were used: PNKP (1:1000, sc-365724 Santa Cruz, Texas, USA; PA5-82263, Invitrogen, Massachusetts, USA), pSTAT1 (1:1000–1:3000, sc-136229, Santa Cruz; and 9167S, Cell Signaling), total STAT1 (1:1000, sc-462,1:3000, Santa Cruz; MA5-15129, 1:2000, Invitrogen), pIRF3 (1:1000, ab76493, Abcam, Cambridge, UK), total IRF3 (1:1000, 11904S, Cell Signaling, Massachusetts, USA), ISG15 (1:1000, sc-166755, Santa Cruz), STING (1:1000, 13647S, Cell Signaling), pTBK1(1:1000, ab109272, Abcam; and MA5-35869, Invitrogen), total TBK1 (1:1000, MA1-20344, Invitrogen), β-actin (1:2000, sc-47778, Santa Cruz), GAPDH (1:3000, NBP2-27103, Novus, Colorado, USA; and MA5-15738-D680, Invitrogen), β-tubulin (1:3000, 926–42211, Li-Cor, Nebraska, USA; and ab6046, Abcam), MTCO1 (1:1000, 459600, Invitrogen), MAVS (1:1000, sc-166583, Santa Cruz), TFAM (1:2000, PA5-29571, Invitrogen), TREX1 (1:1000, 15107S, Cell Signaling; and MA5-34734, Invitrogen), Lamin A/C (1:1000, MA3-1000, Invitrogen), cGAS (1:1000, PA5-76367, Invitrogen; and 15102S, Cell Signaling), Nucleolin (1:2000, ab22758, Abcam), and anti-dsDNA (1:1000, ab27156, Abcam).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Incubation, Cell Harvesting, Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Fractionation