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(A) GSDMD cleavage in THP-1 cells infected with SFTSV (MOI = 1) or stimulated with VbP (2 μM) for 24 h. (B) Knockout of <t>CARD8</t> in THP-1 cells identified with Western blot. (C) Knockout of NLRP3 in THP-1 cells identified with Western blot. (D-F) GSDMD cleavage (D), IL-1β production ( E ) and LDH release ( F ) in THP-1 WT, CARD8 KO and NLRP3 KO infected with SFTSV (MOI = 1) or stimulated with VbP (5 μM) for 24 h. (G) THP-1 cells were infected with SFTSV at different MOIs for 24 h, endogenous CARD8 was detected using N-terminal and C-terminal antibodies with Western blot, respectively. (H) A549 cells were infected with SFTSV at different MOIs for 24 h, endogenous CARD8 was detected using N-terminal and C-terminal antibodies with Western blot, respectively. (I) A549 cells were infected with SFTSV (MOI = 1) at indicated time, endogenous CARD8 was detected using N-terminal and C-terminal antibodies with Western blot, respectively. All data represent three independent experiments and presented as mean±s.d. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns , not significant. For statistical analysis, two-tailed unpaired Student’s t -test in (E, F).

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1) Product Images from "The non-structural protein of SFTSV activates NLRP1 and CARD8 inflammasome through disrupting the DPP9-mediated ternary complex"

Article Title: The non-structural protein of SFTSV activates NLRP1 and CARD8 inflammasome through disrupting the DPP9-mediated ternary complex

Journal: PLOS Pathogens

doi: 10.1371/journal.ppat.1013258

(A) GSDMD cleavage in THP-1 cells infected with SFTSV (MOI = 1) or stimulated with VbP (2 μM) for 24 h. (B) Knockout of CARD8 in THP-1 cells identified with Western blot. (C) Knockout of NLRP3 in THP-1 cells identified with Western blot. (D-F) GSDMD cleavage (D), IL-1β production ( E ) and LDH release ( F ) in THP-1 WT, CARD8 KO and NLRP3 KO infected with SFTSV (MOI = 1) or stimulated with VbP (5 μM) for 24 h. (G) THP-1 cells were infected with SFTSV at different MOIs for 24 h, endogenous CARD8 was detected using N-terminal and C-terminal antibodies with Western blot, respectively. (H) A549 cells were infected with SFTSV at different MOIs for 24 h, endogenous CARD8 was detected using N-terminal and C-terminal antibodies with Western blot, respectively. (I) A549 cells were infected with SFTSV (MOI = 1) at indicated time, endogenous CARD8 was detected using N-terminal and C-terminal antibodies with Western blot, respectively. All data represent three independent experiments and presented as mean±s.d. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns , not significant. For statistical analysis, two-tailed unpaired Student’s t -test in (E, F).

Figure Legend Snippet: (A) GSDMD cleavage in THP-1 cells infected with SFTSV (MOI = 1) or stimulated with VbP (2 μM) for 24 h. (B) Knockout of CARD8 in THP-1 cells identified with Western blot. (C) Knockout of NLRP3 in THP-1 cells identified with Western blot. (D-F) GSDMD cleavage (D), IL-1β production ( E ) and LDH release ( F ) in THP-1 WT, CARD8 KO and NLRP3 KO infected with SFTSV (MOI = 1) or stimulated with VbP (5 μM) for 24 h. (G) THP-1 cells were infected with SFTSV at different MOIs for 24 h, endogenous CARD8 was detected using N-terminal and C-terminal antibodies with Western blot, respectively. (H) A549 cells were infected with SFTSV at different MOIs for 24 h, endogenous CARD8 was detected using N-terminal and C-terminal antibodies with Western blot, respectively. (I) A549 cells were infected with SFTSV (MOI = 1) at indicated time, endogenous CARD8 was detected using N-terminal and C-terminal antibodies with Western blot, respectively. All data represent three independent experiments and presented as mean±s.d. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns , not significant. For statistical analysis, two-tailed unpaired Student’s t -test in (E, F).

Techniques Used: Infection, Knock-Out, Western Blot, Two Tailed Test

(A) Colocalization of NSs, NSs-8A and NLRP1 in HEK293T cells co-transfected with HA-NSs, HA-NSs-8A and V5-NLRP1 for 48 h. Scale bar is 10 μm. (B) Co-IP assay between HA-NSs and Myc-CARD8 in HEK293T cells transfected with the indicated expression vectors for 48 h. (C) Co-IP assay between HA-NSs and Flag-CARD8-FIIND in HEK293T cells transfected with the indicated expression vectors for 48 h. (D) Co-IP assay between Flag-NSs or Flag-NSs-8A and Myc-CARD8 in HEK293T cells transfected with the indicated expression vectors for 48 h. (E) GSDMD cleavage in THP-1 cells infected with lentiviruses expression Flag-NSs-WT or Flag-NSs-8A or carrying vector for 48 h.

Figure Legend Snippet: (A) Colocalization of NSs, NSs-8A and NLRP1 in HEK293T cells co-transfected with HA-NSs, HA-NSs-8A and V5-NLRP1 for 48 h. Scale bar is 10 μm. (B) Co-IP assay between HA-NSs and Myc-CARD8 in HEK293T cells transfected with the indicated expression vectors for 48 h. (C) Co-IP assay between HA-NSs and Flag-CARD8-FIIND in HEK293T cells transfected with the indicated expression vectors for 48 h. (D) Co-IP assay between Flag-NSs or Flag-NSs-8A and Myc-CARD8 in HEK293T cells transfected with the indicated expression vectors for 48 h. (E) GSDMD cleavage in THP-1 cells infected with lentiviruses expression Flag-NSs-WT or Flag-NSs-8A or carrying vector for 48 h.

Techniques Used: Transfection, Co-Immunoprecipitation Assay, Expressing, Infection, Plasmid Preparation

(A) Primary keratinocytes were infected with SFTSV (MOI = 1) or stimulated with VbP (5 μM) for 36 h, endogenous DPP8 and DPP9 were detected with Western blot. (B) THP-1 cells were infected with SFTSV (MOI = 1) or stimulated with VbP (2 μM) for 24 h, endogenous DPP8 and DPP9 were detected with Western blot. (C) A549 cells were infected with different MOI SFTSV for 24 h, endogenous DPP8 and DPP9 were detected with Western blot. (D) A549 cells expressing Tet-HA-NSs were treated with doxycycline (1 μg/ml) for 48 h, and identified with Western blot. (E) A549-Tet-HA-NSs cells were treated with doxycycline (1 μg/ml) for 48 h, endogenous DPP8 and DPP9 were detected with Western blot. (F) THP-1-Tet-HA-NSs cells were treatment with doxycycline (1 μg/ml) for 48 h, endogenous DPP8 and DPP9 were detected with Western blot. (G) A549-Tet-HA-NSs cells were treatment with doxycycline (1 μg/ml) for 48 h and then treated with MG132 (10 μM) or CQ (50 μM) for 6 h before harvest. (H) Co-IP of NLRP1-DPP9 with NSs in HEK293T cells transfected with indicated expression vectors for 48 h. (I) Co-IP of CARD8-DPP9 with NSs in HEK293T cells transfected with indicated expression vectors for 48 h. (J) Co-IP of CARD8-DPP9 with NSs in HEK293T cells transfected with Myc-CARD8 and HA-NSs for 48 h.

Figure Legend Snippet: (A) Primary keratinocytes were infected with SFTSV (MOI = 1) or stimulated with VbP (5 μM) for 36 h, endogenous DPP8 and DPP9 were detected with Western blot. (B) THP-1 cells were infected with SFTSV (MOI = 1) or stimulated with VbP (2 μM) for 24 h, endogenous DPP8 and DPP9 were detected with Western blot. (C) A549 cells were infected with different MOI SFTSV for 24 h, endogenous DPP8 and DPP9 were detected with Western blot. (D) A549 cells expressing Tet-HA-NSs were treated with doxycycline (1 μg/ml) for 48 h, and identified with Western blot. (E) A549-Tet-HA-NSs cells were treated with doxycycline (1 μg/ml) for 48 h, endogenous DPP8 and DPP9 were detected with Western blot. (F) THP-1-Tet-HA-NSs cells were treatment with doxycycline (1 μg/ml) for 48 h, endogenous DPP8 and DPP9 were detected with Western blot. (G) A549-Tet-HA-NSs cells were treatment with doxycycline (1 μg/ml) for 48 h and then treated with MG132 (10 μM) or CQ (50 μM) for 6 h before harvest. (H) Co-IP of NLRP1-DPP9 with NSs in HEK293T cells transfected with indicated expression vectors for 48 h. (I) Co-IP of CARD8-DPP9 with NSs in HEK293T cells transfected with indicated expression vectors for 48 h. (J) Co-IP of CARD8-DPP9 with NSs in HEK293T cells transfected with Myc-CARD8 and HA-NSs for 48 h.

Techniques Used: Infection, Western Blot, Expressing, Co-Immunoprecipitation Assay, Transfection

(A-D) THP-1-Ctl, CARD8 KO, and NLRP3 KO were infected with SFTSV (MOI = 1) for 48 h, the mRNA levels ( A ) of SFSTV S, M, L segments were detected with RT-qPCR; ( B ) cells were stained with SFTSV NP and analyzed with immunofluorescence assays. Scale bar, 100 μm; (C) Expression of SFTSV NP was analyzed with Western blot; (D) Functional titers of SFTSV in Ctl, CARD8-KO and NLRP3-KO THP-1 cells measured by TCID 50 . (E-F) THP-1 cells were infected with SFTSV (MOI = 1) and treated with VbP (2 μM) for 24 h. (E) Expression of SFTSV NP was analyzed with Western blot; ( F ) cells were stained with SFTSV NP and analyzed with immunofluorescence assays. Scale bar, 100 μm. All data represent three independent experiments and presented as mean±s.d. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns , not significant. For statistical analysis, two-tailed unpaired Student’s t -test in (A, D).

Figure Legend Snippet: (A-D) THP-1-Ctl, CARD8 KO, and NLRP3 KO were infected with SFTSV (MOI = 1) for 48 h, the mRNA levels ( A ) of SFSTV S, M, L segments were detected with RT-qPCR; ( B ) cells were stained with SFTSV NP and analyzed with immunofluorescence assays. Scale bar, 100 μm; (C) Expression of SFTSV NP was analyzed with Western blot; (D) Functional titers of SFTSV in Ctl, CARD8-KO and NLRP3-KO THP-1 cells measured by TCID 50 . (E-F) THP-1 cells were infected with SFTSV (MOI = 1) and treated with VbP (2 μM) for 24 h. (E) Expression of SFTSV NP was analyzed with Western blot; ( F ) cells were stained with SFTSV NP and analyzed with immunofluorescence assays. Scale bar, 100 μm. All data represent three independent experiments and presented as mean±s.d. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns , not significant. For statistical analysis, two-tailed unpaired Student’s t -test in (A, D).

Techniques Used: Infection, Quantitative RT-PCR, Staining, Immunofluorescence, Expressing, Western Blot, Functional Assay, Two Tailed Test

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Journal: PLOS Pathogens

Article Title: The non-structural protein of SFTSV activates NLRP1 and CARD8 inflammasome through disrupting the DPP9-mediated ternary complex

doi: 10.1371/journal.ppat.1013258

Figure Lengend Snippet: (A) GSDMD cleavage in THP-1 cells infected with SFTSV (MOI = 1) or stimulated with VbP (2 μM) for 24 h. (B) Knockout of CARD8 in THP-1 cells identified with Western blot. (C) Knockout of NLRP3 in THP-1 cells identified with Western blot. (D-F) GSDMD cleavage (D), IL-1β production ( E ) and LDH release ( F ) in THP-1 WT, CARD8 KO and NLRP3 KO infected with SFTSV (MOI = 1) or stimulated with VbP (5 μM) for 24 h. (G) THP-1 cells were infected with SFTSV at different MOIs for 24 h, endogenous CARD8 was detected using N-terminal and C-terminal antibodies with Western blot, respectively. (H) A549 cells were infected with SFTSV at different MOIs for 24 h, endogenous CARD8 was detected using N-terminal and C-terminal antibodies with Western blot, respectively. (I) A549 cells were infected with SFTSV (MOI = 1) at indicated time, endogenous CARD8 was detected using N-terminal and C-terminal antibodies with Western blot, respectively. All data represent three independent experiments and presented as mean±s.d. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns , not significant. For statistical analysis, two-tailed unpaired Student’s t -test in (E, F).

Article Snippet: Human NLRP1 (HG30111-NY), ASC (HG11175-CY), pro-Casp-1 (HG11148-NF), GSDMD (HG25207-NF), pro-IL-1β (HG10139-NM), CARD8 (HG12619-NM) and DPP9 (HG11418-NF) plasmid and the ASC-GFP (HG11175-ACGLN) lentiviral vector were purchased from Sino Biological (Beijing, China).

Techniques: Infection, Knock-Out, Western Blot, Two Tailed Test

Journal: PLOS Pathogens

Article Title: The non-structural protein of SFTSV activates NLRP1 and CARD8 inflammasome through disrupting the DPP9-mediated ternary complex

doi: 10.1371/journal.ppat.1013258

Figure Lengend Snippet: (A) Colocalization of NSs, NSs-8A and NLRP1 in HEK293T cells co-transfected with HA-NSs, HA-NSs-8A and V5-NLRP1 for 48 h. Scale bar is 10 μm. (B) Co-IP assay between HA-NSs and Myc-CARD8 in HEK293T cells transfected with the indicated expression vectors for 48 h. (C) Co-IP assay between HA-NSs and Flag-CARD8-FIIND in HEK293T cells transfected with the indicated expression vectors for 48 h. (D) Co-IP assay between Flag-NSs or Flag-NSs-8A and Myc-CARD8 in HEK293T cells transfected with the indicated expression vectors for 48 h. (E) GSDMD cleavage in THP-1 cells infected with lentiviruses expression Flag-NSs-WT or Flag-NSs-8A or carrying vector for 48 h.

Techniques: Transfection, Co-Immunoprecipitation Assay, Expressing, Infection, Plasmid Preparation

Journal: PLOS Pathogens

Article Title: The non-structural protein of SFTSV activates NLRP1 and CARD8 inflammasome through disrupting the DPP9-mediated ternary complex

doi: 10.1371/journal.ppat.1013258

Figure Lengend Snippet: (A) Primary keratinocytes were infected with SFTSV (MOI = 1) or stimulated with VbP (5 μM) for 36 h, endogenous DPP8 and DPP9 were detected with Western blot. (B) THP-1 cells were infected with SFTSV (MOI = 1) or stimulated with VbP (2 μM) for 24 h, endogenous DPP8 and DPP9 were detected with Western blot. (C) A549 cells were infected with different MOI SFTSV for 24 h, endogenous DPP8 and DPP9 were detected with Western blot. (D) A549 cells expressing Tet-HA-NSs were treated with doxycycline (1 μg/ml) for 48 h, and identified with Western blot. (E) A549-Tet-HA-NSs cells were treated with doxycycline (1 μg/ml) for 48 h, endogenous DPP8 and DPP9 were detected with Western blot. (F) THP-1-Tet-HA-NSs cells were treatment with doxycycline (1 μg/ml) for 48 h, endogenous DPP8 and DPP9 were detected with Western blot. (G) A549-Tet-HA-NSs cells were treatment with doxycycline (1 μg/ml) for 48 h and then treated with MG132 (10 μM) or CQ (50 μM) for 6 h before harvest. (H) Co-IP of NLRP1-DPP9 with NSs in HEK293T cells transfected with indicated expression vectors for 48 h. (I) Co-IP of CARD8-DPP9 with NSs in HEK293T cells transfected with indicated expression vectors for 48 h. (J) Co-IP of CARD8-DPP9 with NSs in HEK293T cells transfected with Myc-CARD8 and HA-NSs for 48 h.

Techniques: Infection, Western Blot, Expressing, Co-Immunoprecipitation Assay, Transfection

Journal: PLOS Pathogens

Article Title: The non-structural protein of SFTSV activates NLRP1 and CARD8 inflammasome through disrupting the DPP9-mediated ternary complex

doi: 10.1371/journal.ppat.1013258

Figure Lengend Snippet: (A-D) THP-1-Ctl, CARD8 KO, and NLRP3 KO were infected with SFTSV (MOI = 1) for 48 h, the mRNA levels ( A ) of SFSTV S, M, L segments were detected with RT-qPCR; ( B ) cells were stained with SFTSV NP and analyzed with immunofluorescence assays. Scale bar, 100 μm; (C) Expression of SFTSV NP was analyzed with Western blot; (D) Functional titers of SFTSV in Ctl, CARD8-KO and NLRP3-KO THP-1 cells measured by TCID 50 . (E-F) THP-1 cells were infected with SFTSV (MOI = 1) and treated with VbP (2 μM) for 24 h. (E) Expression of SFTSV NP was analyzed with Western blot; ( F ) cells were stained with SFTSV NP and analyzed with immunofluorescence assays. Scale bar, 100 μm. All data represent three independent experiments and presented as mean±s.d. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns , not significant. For statistical analysis, two-tailed unpaired Student’s t -test in (A, D).

Techniques: Infection, Quantitative RT-PCR, Staining, Immunofluorescence, Expressing, Western Blot, Functional Assay, Two Tailed Test

(A, B) Transient knockdown of AIM2, NLRP1, NLRP3, CARD8, CASP1, and CASP4 by siRNA transfection in primary MDMs. Knock-down efficiency was determined by RT-qPCR at day 2 post-transfection, and represented as relative mRNA expression of individual targets compared to scramble siControl (A) , and protein level analyzed by immunoblotting (B) . MDMs were infected with Lai∆envGFP/G (MOI 1) for 3 days in the absence (DMSO) or presence of EFV (C–G) . Culture supernatants were harvested at 3 dpi and analyzed for p24 Gag (C) , IL-1β (D) , and IP-10 (E) secretion by ELISA. (F) Bioactive type I IFN in the culture supernatants from infected MDMs at 3 dpi quantified using interferon bioassay. The values in panels B-E were normalized to that of siControl-transfected and HIV-1-infected cells in each donor and reported as relative expression. (G) Quantitation of LDH release in culture supernatants was measured using the CytoTox cytotoxicity assay, followed by subtraction of background OD from culture media, and normalized to EFV-treatment for each siRNA transduction per donor. The means ± SEM are shown, and each symbol represents an independent donor. Statistical significance was determined by two-way ANOVA followed by Dunnett’s post-test comparing HIV-infected MDMs from siControl to indicated specific siRNA (D, G) . P -values: **** < 0.0001; * < 0.05; no symbol: not significant ( p ≥ 0.05). The data underlying this figure can be found in .

Journal: PLOS Biology

Article Title: Expression of intron-containing HIV-1 RNA induces NLRP1 inflammasome activation in myeloid cells

doi: 10.1371/journal.pbio.3003320

Figure Lengend Snippet: (A, B) Transient knockdown of AIM2, NLRP1, NLRP3, CARD8, CASP1, and CASP4 by siRNA transfection in primary MDMs. Knock-down efficiency was determined by RT-qPCR at day 2 post-transfection, and represented as relative mRNA expression of individual targets compared to scramble siControl (A) , and protein level analyzed by immunoblotting (B) . MDMs were infected with Lai∆envGFP/G (MOI 1) for 3 days in the absence (DMSO) or presence of EFV (C–G) . Culture supernatants were harvested at 3 dpi and analyzed for p24 Gag (C) , IL-1β (D) , and IP-10 (E) secretion by ELISA. (F) Bioactive type I IFN in the culture supernatants from infected MDMs at 3 dpi quantified using interferon bioassay. The values in panels B-E were normalized to that of siControl-transfected and HIV-1-infected cells in each donor and reported as relative expression. (G) Quantitation of LDH release in culture supernatants was measured using the CytoTox cytotoxicity assay, followed by subtraction of background OD from culture media, and normalized to EFV-treatment for each siRNA transduction per donor. The means ± SEM are shown, and each symbol represents an independent donor. Statistical significance was determined by two-way ANOVA followed by Dunnett’s post-test comparing HIV-infected MDMs from siControl to indicated specific siRNA (D, G) . P -values: **** < 0.0001; * < 0.05; no symbol: not significant ( p ≥ 0.05). The data underlying this figure can be found in .

Article Snippet: At day 2 post-transfection, cells were either infected with HIV-1 (as described above), or stimulated as follows to determine functional knockdown: AIM2 (cells were treated with ultra-pure LPS (100 ng/m, Invivogen) for 2 h, followed by transfection with linearized DNA (1 μg/mL) for 4 h); NLRP1 and CARD8 (cells were primed with Pam3CSK4 (0.5 μg/mL, InvivoGen) for 4 h, followed by stimulation with Val-boroPro (10 μM, InvivoGen) for 24 h; NLRP3 and caspase-1 (cells were primed with ATP (5 mM, Thermo Scientific) for 6 h, followed by activation with nigericin (10 μM) for 60 min; Caspase-4 (cells were transfected with ultra-pure LPS (5 μg/ml, InvivoGen) for 6 hrs; UNC93B1 (cells were treated with Resiquimod (5 μg/ml, Invivogen) for 24 h. At the indicated timepoints, culture supernatants were harvested for IL-1β and IP-10 ELISA, and cells were lysed for RNA or protein analysis.

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Expressing, Western Blot, Infection, Enzyme-linked Immunosorbent Assay, Bioassay, Quantitation Assay, Cytotoxicity Assay, Transduction

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