Journal: Nature immunology

Article Title: NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component

doi: 10.1038/ni.3333

Figure Lengend Snippet: NLRP3 inflammasome activation is blocked during mitosis. ( a ) NEK7-NLRP3 association in LPS-primed J774A.1 mitotic or interphase cells, with or without HA-NEK7 overexpression, analyzed by immunoprecipitation and immunoblot. ( b ) J774A.1 cells were primed with LPS and stimulated with nigericin together with fluorescent FAM-FLICA-caspase-1 probe specific for activated caspase-1. Flow cytometric analysis of cells containing activated caspase-1 (left); their frequency among mitotic and interphase cells is graphed (right). ( c-e ) J774A.1 cells were arrested at the G2/M phase border by incubation with RO-3306, then released from arrest for different time periods before analysis. ( c ) Cells were treated with RO-3306 for 20 h, with LPS added during the last 4 h. Release was in fresh media without RO-3306 and LPS for the indicated times. Flow cytometric analysis of phosphorylated histone H3-positive mitotic cells (left); the percentage of mitotic cells is indicated. The interaction between endogenous NEK7 and NLRP3 was analyzed by immunoprecipitation and immunoblot (right). ( d ) ELISA analysis of IL-1β in the culture supernatants of J774A.1 cells primed with LPS and stimulated with nigericin. Release from RO-3306 arrest was 1 h before (teal) or concurrent with (blue) nigericin stimulation. ( e ) ELISA analysis of IL-1β in the culture supernatants of J774A.1 cells stably overexpressing NEK7 or NEK7 K64M primed with LPS and stimulated with nigericin. RO-3306 release was at the time nigericin was added. * P ≤0.05; ** P ≤0.01; *** P ≤0.001 (unpaired, two-tailed Student's t test). For b, d , and e , the means of triplicate samples are plotted. Results are representative of two independent experiments.

Article Snippet: Ultra-pure LPS, MALP-2 and R848 were obtained from Enzo Life Sciences; for stimulation of human monocytes, LPS (O111:B4) was from List Biologicals.

Techniques: Activation Assay, Over Expression, Immunoprecipitation, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Stable Transfection, Two Tailed Test

Journal: eLife

Article Title: Ebola virus sequesters IRF3 in viral inclusion bodies to evade host antiviral immunity

doi: 10.7554/eLife.88122

Figure Lengend Snippet: ( A ) HEK293 cells were transfected with the indicated plasmids for 24 hr, and the cells were infected with or without Sendai virus (SeV) at an MOI of 2 for another 12 hr. The mRNA level of IFN-β was quantified by quantitative RT-PCR (qRT-PCR). Differences between the two groups were evaluated by a two-sided unpaired Student’s t -test. The data are presented as the means ± standard error of the mean (SEM; n=3; *p < 0.05, **p < 0.01, ***p < 0.001). ( B ) HEK293 cells were cotransfected with the firefly luciferase reporter plasmid pGL3-IFN-β-Luc, the Renilla luciferase control plasmid pRL-TK, and viral protein expression plasmids (0.0625 μg of pCAGGS-NP, 0.0625 μg of pCAGGS-VP35, 0.0375 μg of pCAGGS-VP30, and 0.5 μg of pCAGGS-L) for 24 hr, and the cells were infected with or without SeV at an MOI of 2 for another 12 hr. The luciferase activities were then analyzed. The data were analyzed to determine the fold induction by normalizing the firefly luciferase activity to the Renilla luciferase activity. Empty plasmid without SeV infection was used as a control, and the corresponding data point was set to 100%. Differences between the two groups were evaluated using a two-sided unpaired Student’s t -test. The data are presented as the means ± SEM (n=3; ns, not significant, *p < 0.05, ***p < 0.001). ( C ) Wild-type (WT) and IRF3 -depleted (IRF3 −/− ) HeLa cells were transfected with or without pCASSG-NP, pCASSG-VP35, pCASSG-VP30, and pCASSG-L plasmids for 36 hr and then treated with or without 5 μg/ml poly(I:C) for 12 hr. The mRNA level of IFN-β was quantified by qRT-PCR. Differences between the two groups were evaluated using a two-sided unpaired Student’s t -test. The data are presented as the means ± SEM (n=3; ns, not significant, *p < 0.05). ( D–F ) Wild-type (WT) and IRF3 -depleted (IRF3 −/− ) HeLa cells were transfected with or without pCAGGS-VP35 or pCASSG-NP, pCASSG-VP35, pCASSG-VP30, and pCASSG-L plasmids for 36 hr, and the cells were infected with or without SeV at an MOI of 5 for another 12 hr. The mRNA level of CXCL10 ( D ), ISG15 ( E ), and ISG56 ( F ) was quantified by qRT-PCR. Differences between the two groups were evaluated using a two-sided unpaired Student’s t -test. The data are presented as the means ± SEM (n=3; *p < 0.05, **p < 0.01, ***p < 0.001). ( G ) Wild-type (WT) and IRF3 -knockout (IRF3 −/− ) HeLa cells were transfected with the Ebola virus (EBOV) minigenome (p0), pGL3-promoter and Myc-vector, Myc-IRF3 or Myc-IRF3/5D plasmids for 96 hr. The amounts of transcription- and replication-competent virus-like particles (trVLPs) were determined by a luciferase activity assay (left panel). Differences between the two groups were evaluated by a two-sided unpaired Student’s t -test. The data are presented as the means ± SEM (n=3; ns, not significant, ***p < 0.001). ( H ) Wild-type (WT) and IRF3 -knockout (IRF3 −/− ) HeLa cells were infected with live EBOV (MOI = 0.1). The cell culture supernatants were collected on the indicated days post infection (dpi), and the viral titers were quantified as TCID 50 by a plaque assay. Differences between the two groups were evaluated using a two-sided unpaired Student’s t -test. The data are presented as the means ± SEM (n=3; ns, not significant). Figure 7—source data 1. Numerical data for . Figure 7—source data 2. Numerical data for . Figure 7—source data 3. Numerical data for . Figure 7—source data 4. Numerical data for . Figure 7—source data 5. Numerical data for . Figure 7—source data 6. Numerical data for . Figure 7—source data 7. Numerical data for . Figure 7—source data 8. Numerical data for .

Article Snippet: Briefly, HEK293 cells (1 × 10 5 cells per well in a 24-well plate) were cotransfected with the indicated amount of pCAGGS-NP (62.5 ng)/pCAGGS-VP35 (62.5 ng)/pCAGGS-VP30 (37.5 ng)/pCAGGS-L (500 ng), 200 ng of the IFN-β reporter plasmid (Promega, USA) and 4 ng of Renilla luciferase plasmid.

Techniques: Transfection, Infection, Virus, Quantitative RT-PCR, Luciferase, Plasmid Preparation, Expressing, Activity Assay, Knock-Out, Cell Culture, Plaque Assay

Journal: eLife

Article Title: Ebola virus sequesters IRF3 in viral inclusion bodies to evade host antiviral immunity

doi: 10.7554/eLife.88122

Figure Lengend Snippet:

Article Snippet: Briefly, HEK293 cells (1 × 10 5 cells per well in a 24-well plate) were cotransfected with the indicated amount of pCAGGS-NP (62.5 ng)/pCAGGS-VP35 (62.5 ng)/pCAGGS-VP30 (37.5 ng)/pCAGGS-L (500 ng), 200 ng of the IFN-β reporter plasmid (Promega, USA) and 4 ng of Renilla luciferase plasmid.

Techniques: Virus, Recombinant, Plasmid Preparation, Synthesized, Sequencing, SYBR Green Assay, Real-time Polymerase Chain Reaction, In Situ, Reporter Assay, Software, Staining, Immunofluorescence

Journal: Cancer research

Article Title: KDM5B is essential for the hyper-activation of PI3K/AKT signaling in prostate tumorigenesis

doi: 10.1158/0008-5472.CAN-20-0505

Figure Lengend Snippet: (A) Left panel, western blotting analysis showing the protein levels of KDM5B, pAKT, P110α, and P85 in LNCaP KDM5B-KO and the parental control human PCa cells. Right panel, quantification analysis of pAKT, P110α, and P85 in LNCaP KDM5B-KO and the control cells. (B) A comparison of the cell proliferation between LNCaP KDM5B-KO and the control cells. (C) Immunofluorescence (IF) images and analysis of PIP3 in LNCaP KDM5B-KO cells. Left panel, IF images showing the levels and membrane localization of PIP3 in LNCaP KDM5B-KO cells and the control cells. Right panel, quantification of the fluorescence intensities of PIP3 in LNCaP KDM5B-KO and the control cells. Error bars represent means ± SD (20 cells/group). (D) RNA-Seq peaks showing the changes of KDM5B, IRS1, PIK3CA, and PIK3R1 expression between LNCaP KDM5B-KO and the control cells. (E) Quantitative RT-PCR analysis to show the relative mRNA levels of IRS1, PIK3CA, and PIK3R1 in LNCaP KDM5B-KO cells. (F) Top panel, western blotting analysis showing the protein levels of KDM5B, P110α and P85 in LNCaP KDM5B-KO and the control cells at indicated time points in CHX chase experiments. Bottom panel, quantification of protein remaining for P110α and P85 at indicated time points in KDM5B-KO and the control cells.

Article Snippet: For PIK3CA promoter reporter assay, cells were transfected with 200 ng empty pLightSwitch_Prom reporter vector, 50 ng, 100 ng or 200 ng of PIK3CA -promoter-Luc reporter plasmids (Switchgear Genomics, S706543).

Techniques: Western Blot, Immunofluorescence, Fluorescence, RNA Sequencing Assay, Expressing, Quantitative RT-PCR

Journal: Cancer research

Article Title: KDM5B is essential for the hyper-activation of PI3K/AKT signaling in prostate tumorigenesis

doi: 10.1158/0008-5472.CAN-20-0505

Figure Lengend Snippet: (A) A schematic showing the positions of 5 amplicons relative to the PIK3CA transcription start site (TSS). (B) ChIP analysis of KDM5B levels at the indicated regions near PIK3CA TSS in LNCaP KDM5B-KO and the parental control cells. (C) ChIP analysis of H3K4me3 levels at the indicated regions near PIK3CA TSS in LNCaP KDM5B-KO and the control cells. (D) Top panel, a schematic showing that KDM5B regulates the transcription of PIK3CA through affecting promoter activities. Bottom panel, comparisons of luciferase activities of the PIK3CA promoter between LNCaP KDM5B-KO cells and the parental control cells. (E) Luciferase activities showing the effects of KDM5B restoration on the PIK3CA promoter activities in LNCaP KDM5B-KO cells.

Article Snippet: For PIK3CA promoter reporter assay, cells were transfected with 200 ng empty pLightSwitch_Prom reporter vector, 50 ng, 100 ng or 200 ng of PIK3CA -promoter-Luc reporter plasmids (Switchgear Genomics, S706543).

Techniques: Luciferase