Journal: Immunity

Article Title: Germ-cell specific inflammasome component NLRP14 negatively regulates cytosolic nucleic acid sensing to promote fertilization

doi: 10.1016/j.immuni.2017.03.020

Figure Lengend Snippet: (A) Schematic diagram outlining signaling pathway components involved in cytosolic nucleic acid sensing. (B–D) 293T cells were cotransfected with Myc-NLRP14 and RIG-I-Flag, MAVS-Flag, STING-HA, TBK1-Flag, TRIF-Flag, IRF3-Flag, or untagged-IRF7 along with the luciferase gene under control of the ISRE or NFκB promoter. Shown are relative luciferase units (RLU) normalized to Renilla luciferase expression. (E) 293T cells were cotransfected with TBK1-Flag and decreasing amounts of Myc-NLRP14. IP10, IFNβ, and NLRP14 mRNA levels were determined by real-time PCR 24h post transfection. RT-PCR data were normalized to GAPDH mRNA. **P<0.01, Student’s T-test. Error bars indicate SD. Data are representative of at least three independent experiments. (See also Supplemental Figure S1 and S3)

Article Snippet: Human IP10, IFNβ, IRF7, ISG15, and GAPDH genes were amplified using the following primer pairs: IP10 : 5′-GGCCATCAAGAATTTACTGAAAGCA-3′ and 5′-TCTGTGTGGTCCATCCTTGGAA-3′ IFNβ : 5′-TCTGGCACAACAGGTAGTAGGC-3′ and 5′-GAGAAGCACAACAGGAGAGCAA-3′ IRF7 : 5′-TGGTCCTGGTGAAGCTGGAA-3′ and 5′-GATGTCGTCATAGAGGCTGTTGG-3′ ISG15 : 5′-GAGAGGCAGCGAACTCATCT-3′ and 5′-CTTCAGCTCTGACACCGACA-3′ GAPDH : 5′- ACCACAGTCCATGCCATCAC -3′ and 5′- TCCACCACCCTGTTGCTGTA -3′ The qRT-PCR primers for NLRP14 and NLRP4 were purchased from Origene.

Techniques: Luciferase, Control, Expressing, Real-time Polymerase Chain Reaction, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: Immunity

Article Title: Germ-cell specific inflammasome component NLRP14 negatively regulates cytosolic nucleic acid sensing to promote fertilization

doi: 10.1016/j.immuni.2017.03.020

Figure Lengend Snippet: (A) 293T-STING-HA-NLRP14-CRISPR cells were transfected with cGAS-Flag, or decreasing doses of 2′3′-cGAMP (30, 15, 7.5 and 1.5 μg/ml) along with ISRE or IFNβ promoters. Shown are relative luciferase units (RLU) normalized to Renilla luciferase expression. (B) Parental and 293T-NLRP14-CRISPR cells were stimulated with B-DNA (0.06 or 0.03 μg/ml), PolyI:C (25, 12.5, and 2.5 μg/ml), or infected with NDV-GFP (moi of 20, 4, or 0.8). 24h after treatment, levels of ISRE inducing cytokines in supernatants were determined using 293T-ISRE-B8 cells. (C) Rhodamine-labeled B-DNA (indicated as B-DNA Rho) was transfected into parental (WT) and 293T-NLRP14-CRISPR (KO) cells, and observed by fluorescence microscopy. (D) Parental and 293T-NLRP14-CRISPR cells were stimulated with indicated ligands (0.06 μg/ml of B-DNA and NDV-GFP at the m.o.i of 10), and levels of IP10, IFNβ, IRF7 and ISG15 mRNA were determined by RT-PCR 6h post transfection of B-DNA and 15h post NDV inoculation. (E) Parental and 293T-NLRP14-CRISPR cells were transfected with RIG-I-Flag, MAVS-Flag, TBK1-Flag, or STING-HA, and IP10 and IFNβ mRNA levels were determined by RT-PCR at 24h post transfection. RT-PCR data were normalized to GAPDH mRNA. **P<0.01, ***P<0.001, Student’s T-test. Error bars indicate SD. Data are representative of at least three independent experiments. Black and red bars indicate parental and CRISPR-targeted cells, respectively. (See also Supplemental Figures S2 and S4)

Article Snippet: Human IP10, IFNβ, IRF7, ISG15, and GAPDH genes were amplified using the following primer pairs: IP10 : 5′-GGCCATCAAGAATTTACTGAAAGCA-3′ and 5′-TCTGTGTGGTCCATCCTTGGAA-3′ IFNβ : 5′-TCTGGCACAACAGGTAGTAGGC-3′ and 5′-GAGAAGCACAACAGGAGAGCAA-3′ IRF7 : 5′-TGGTCCTGGTGAAGCTGGAA-3′ and 5′-GATGTCGTCATAGAGGCTGTTGG-3′ ISG15 : 5′-GAGAGGCAGCGAACTCATCT-3′ and 5′-CTTCAGCTCTGACACCGACA-3′ GAPDH : 5′- ACCACAGTCCATGCCATCAC -3′ and 5′- TCCACCACCCTGTTGCTGTA -3′ The qRT-PCR primers for NLRP14 and NLRP4 were purchased from Origene.

Techniques: CRISPR, Transfection, Luciferase, Expressing, Infection, Labeling, Fluorescence, Microscopy, Reverse Transcription Polymerase Chain Reaction

Journal: Immunity

Article Title: Germ-cell specific inflammasome component NLRP14 negatively regulates cytosolic nucleic acid sensing to promote fertilization

doi: 10.1016/j.immuni.2017.03.020

Figure Lengend Snippet: (A) Schematic of human NLRP14 and deletion mutants generated. Pyrin domain (PYD); NAIP, CIITA, HET-E, and TP1 domain (NACHT); Leucine rich repeat (LRR). 293T cells were cotransfected with TBK1 and NLRP14 variants along with IFNβ-luc or NFκB-luc and monitored for promoter activity (RLU; B) or subjected to immunoblotting with appropriate antibodies (C). (D) 293T cells were transfected with full length, Δ1-345 or Δ531-1093 NLRP14 and interaction with endogenous TBK1 monitored by immunoprecipitation 48h post transfection. (E) 293T cells were cotransfected with TBK1 and NLRP14 variants and IP10 and IFNβ mRNA levels were determined by RT-PCR 24h post transfection. **P<0.01, Student’s T-test. Error bars indicate SD. Data are representative of at least two independent experiments. (See also Supplemental Figure S6)

Article Snippet: Human IP10, IFNβ, IRF7, ISG15, and GAPDH genes were amplified using the following primer pairs: IP10 : 5′-GGCCATCAAGAATTTACTGAAAGCA-3′ and 5′-TCTGTGTGGTCCATCCTTGGAA-3′ IFNβ : 5′-TCTGGCACAACAGGTAGTAGGC-3′ and 5′-GAGAAGCACAACAGGAGAGCAA-3′ IRF7 : 5′-TGGTCCTGGTGAAGCTGGAA-3′ and 5′-GATGTCGTCATAGAGGCTGTTGG-3′ ISG15 : 5′-GAGAGGCAGCGAACTCATCT-3′ and 5′-CTTCAGCTCTGACACCGACA-3′ GAPDH : 5′- ACCACAGTCCATGCCATCAC -3′ and 5′- TCCACCACCCTGTTGCTGTA -3′ The qRT-PCR primers for NLRP14 and NLRP4 were purchased from Origene.

Techniques: Generated, Activity Assay, Western Blot, Transfection, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction