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

Journal: Journal of the American Society of Nephrology : JASN

Article Title: IL-6 Trans–Signaling Links Inflammation with Angiogenesis in the Peritoneal Membrane

doi: 10.1681/ASN.2015101169

Figure Lengend Snippet: The role of STAT3 in SP4–mediated VEGF induction by the IL-6 + sIL-6R complex. (A and B) Kinetics of STAT3 and SP4 mRNA induction in HPMCs treated with IL-6 + sIL-6R (both at 100 ng/ml). *P<0.05 versus control cells at each time point (n=4). (C–F) Effect of STAT3 silencing on STAT3, SP4, and VEGF expression. Cells were transiently transfected with either STAT3 siRNA or scrambled (scramb.) siRNA and then, stimulated with IL-6 + sIL-6R (both at 100 ng/ml) for 9 hours. Cells were assessed for (C) STAT3 protein expression by Western blotting and mRNA expression by quantitative PCR for (D) STAT3, (E) SP4, and (F) VEGF. In C, a representative immunoblot is presented together with quantified data from four independent experiments. STAT3 protein expression was normalized per that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). *P<0.05 versus cells stimulated with IL-6 + sIL-6R in the absence of siRNA (n=4).

Article Snippet: Cell extracts were prepared as described, 58 electrophoresed on SDS-PAGE, and Western blotted using antibodies against STAT3 and SP4 (Santa Cruz Biotechnology), GAPDH (Hytest, Turku, Finland), and appropriate secondary peroxidase–conjugated IgG (Dianova, Hamburg, Germany).

Techniques: Control, Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction