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

Journal: Scientific Reports

Article Title: Nucleocapsid Interacts with NPM1 and Protects it from Proteolytic Cleavage, Enhancing Cell Survival, and is Involved in PEDV Growth

doi: 10.1038/srep39700

Figure Lengend Snippet: Western blot analysis of nuclear and cytoplasmic fractions of PEDV-infected Vero E6 cells with an anti-GAPDH mAb ( A ), anti-PCNA mAb ( B ), and anti-N mAb ( C ). ( A,B and C ) Nuc, nuclear fraction of PEDV-infected cells; Cyt, cytoplasmic fraction of PEDV-infected cells. ( C ) Moc, mock-infected cells; Inf, PEDV-infected cells. The arrowheads indicate purified bands that are the same sizes as GAPDH ( A ), PCNA ( B ), and N ( C ) proteins. ( D ) Western blot analysis of N protein in nuclear and cytoplasmic fractions of PEDV-infected Vero E6 cells at different times with an anti-GAPDH mAb, anti-PCNA mAb, and anti-N mAb. Nuc, nuclear fraction of PEDV-infected cells; Cyt, cytoplasmic fraction of PEDV-infected cells.

Article Snippet: The membrane was soaked in blocking buffer (PBS containing 5% nonfat milk) for 2 h and then reacted with the indicated antibodies: mouse anti-GAPDH mAb (1:10,000) (G8795; Sigma), mouse anti-PCNA mAb (1:200) (BM0104; BOSTER), mouse anti-N mAb (1:500), mouse anti-GST mAb (1:1000) (AG768; Beyotime), mouse anti-actin mAb (1:5000) (A5441; Sigma), mouse anti-GFP mAb (1:10,000) (66002–1–1 g; Proteintech), rabbit anti-Flag mAb (1:2000) (20543–1-AP; Proteintech), or mouse anti Myc mAb (1:2000) (66004–1–1 g; Proteintech), rabbit anti-caspase-3 mAb (1:1000) (AC030; Beyotime).

Techniques: Western Blot, Infection, Purification

Journal: Scientific Reports

Article Title: Nucleocapsid Interacts with NPM1 and Protects it from Proteolytic Cleavage, Enhancing Cell Survival, and is Involved in PEDV Growth

doi: 10.1038/srep39700

Figure Lengend Snippet: ( A ) The NPM1 fusion protein as a marker of the nucleolus is colored red. The nucleolar localization of N in transfected cells was clearly observed at 42–42.5 hpt. As shown, a small amount of N was observed in the nucleolus at 42 hpt (t = 30 min). N protein accumulated continuously in the nucleolus of transfected cells until t = 60 min and was exported from the nucleolus at t = 61–65 min. This figure shows snapshots of the cells from the time-lapse movie ( in the ). Data are representative of one of three independent experiments. Real-time visualization of the kinetics of the nucleolar localization of N protein indicated that the process was rapid, taking only 30 min in total. ( B ) Knockdown of NPM1 protein levels following siRNA treatment. Vero E6 cells transfected with no siRNA (Mock), scrambled siRNA (siScr), left untreated (No treat) or with different concentrations (mM) of siRNAs targeting NPM1 (siNPM1) (as indicated at the top of each lane) were harvested 48 hpt. Endogenous NPM1 protein levels were detected by immunoblotting using antibodies directed against the indicated proteins. ( C and D ) Western blot analysis of Myc-N protein in nuclear and cytoplasmic fractions of NPM1-knockdown cells ( C ) or Ectopic NPM1-overexpression cells ( D ) at 48 hpt with anti-GAPDH mAb, anti-PCNA mAb, anti-NPM1 mAb, anti-Myc mAb and anti-Flag mAb. Nuc, nuclear fraction; Cyt, cytoplasmic fraction; cell, whole cells. Densitometric data for Nuc/Cell (Myc-N) from three independent experiments are expressed as mean ± SD.

Article Snippet: The membrane was soaked in blocking buffer (PBS containing 5% nonfat milk) for 2 h and then reacted with the indicated antibodies: mouse anti-GAPDH mAb (1:10,000) (G8795; Sigma), mouse anti-PCNA mAb (1:200) (BM0104; BOSTER), mouse anti-N mAb (1:500), mouse anti-GST mAb (1:1000) (AG768; Beyotime), mouse anti-actin mAb (1:5000) (A5441; Sigma), mouse anti-GFP mAb (1:10,000) (66002–1–1 g; Proteintech), rabbit anti-Flag mAb (1:2000) (20543–1-AP; Proteintech), or mouse anti Myc mAb (1:2000) (66004–1–1 g; Proteintech), rabbit anti-caspase-3 mAb (1:1000) (AC030; Beyotime).

Techniques: Marker, Transfection, Knockdown, Western Blot, Over Expression

Journal: Scientific Reports

Article Title: Nucleocapsid Interacts with NPM1 and Protects it from Proteolytic Cleavage, Enhancing Cell Survival, and is Involved in PEDV Growth

doi: 10.1038/srep39700

Figure Lengend Snippet: ( A and B ) N protein binding prevents NPM1 proteolytic cleavage. Vero E6 cells were transfected with pMyc-N or empty vector for 24 h and then treated with or without 100 μM of Ac-DEVD-CHO (caspase-3 inhibitor) for 6 h. The cells were treated with or without 250 nm of STS for 18 h. The western blots were probed for freshly extracted proteins with antibodies against NPM1, Myc, GAPDH and caspase-3. Verification of Myc-N induction and equal sample loading are shown by anti-Myc and anti-GAPDH mAbs. CF, cleavage fragment. ( C ) N protein enhances the antiapoptotic effect of NPM1. Vero E6 cells were transfected with pMyc-N or empty vector for 30 h and then treated with or without 250 nm of STS for 18 h. Genomic DNA was loaded on to a 2% agarose gel. Verification of Myc-N induction and equal sample loading are shown by anti-Myc and anti-GAPDH mAbs. ( D ) Vero E6 cells were transfected with pMyc-N or empty vector for 30 h and then treated with or without 250 nm of STS for 18 h, then TUNEL and DAPI staining to examine the apoptotic cell death. Statistical results represent means ± SD of apoptotic cell counts from six different fields (right).

Article Snippet: The membrane was soaked in blocking buffer (PBS containing 5% nonfat milk) for 2 h and then reacted with the indicated antibodies: mouse anti-GAPDH mAb (1:10,000) (G8795; Sigma), mouse anti-PCNA mAb (1:200) (BM0104; BOSTER), mouse anti-N mAb (1:500), mouse anti-GST mAb (1:1000) (AG768; Beyotime), mouse anti-actin mAb (1:5000) (A5441; Sigma), mouse anti-GFP mAb (1:10,000) (66002–1–1 g; Proteintech), rabbit anti-Flag mAb (1:2000) (20543–1-AP; Proteintech), or mouse anti Myc mAb (1:2000) (66004–1–1 g; Proteintech), rabbit anti-caspase-3 mAb (1:1000) (AC030; Beyotime).

Techniques: Protein Binding, Transfection, Plasmid Preparation, Western Blot, Agarose Gel Electrophoresis, TUNEL Assay, Staining