Fig 1: The role of SFTSV NSs in the activation of the NLRP1 and CARD8 inflammasome.(A) ASC-caspase-1-pro-IL-1β HEK293T cells were transfected with indicated expression vector for 36 h. Supernatants were analyzed for IL-1β with ELISA. (B) ASC-caspase-1-pro-IL-1β HEK293T cells were transfected with plasmids NSs, production of p17 and GSDMD processing were detected with Western blot. (C-D) ASC specks fluorescence microscopy images (C) and quantification (D) of A549-HA-NLRP1-Flag-ASC-GFP cells transfected NSs-HA for 24 h. Scale bar, 100 μm. (E-F) GSDMD cleavage (E) and IL-1β (F) production in primary keratinocytes expressing Tet-HA-NSs and treated with doxycycline (1 μg/ml) for 48 h. (G-H) GSDMD cleavage (G) and IL-1β (H) production in THP-1 cells expressing Tet-HA-NSs and treated with doxycycline (1 μg/ml) for 48 h. 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, F, H).
Fig 2: SFTSV induces NLRP1 inflammasome activation in primary keratinocytes.(A-B) ASC specks fluorescence microscopy images (A) and quantification (B) of A549-HA-NLRP1-Flag-ASC-GFP cells infected with SFTSV or without SFTSV (MOI = 0.5) for 24 h. Scale bar, 100 μm. (C-D) Detection of SFTSV NP (C) and IL-β production (D) in primary keratinocytes infected with SFTSV at different MOIs or stimulated with VbP (2 μM) for 24 h. (E-F) GSDMD cleavage (E) and LDH release (F) in primary keratinocytes infected with SFTSV (MOI = 1) or stimulated with VbP (5 μM) for 36 h. (G) Expression of NLRP1 in primary keratinocytes treated with lentivirus-mediated CRISPR-Cas9 and NLRP1-specific single-guide RNA (sgRNA) or the non-targeting sgRNA control. (H) IL-1β production in wild-type or NLRP1-deficient primary keratinocytes infected with SFTSV (MOI = 1) or stimulated with VbP (2 μM) for 24 h. (I) Primary keratinocytes were infected with SFTSV at different MOIs for 24 h, endogenous NLRP1 was detected using N-terminal and C-terminal antibodies with Western blot, respectively. (J-K) Primary keratinocytes were infected with SFTSV (MOI = 1) and treated with MG132 (2 μM) for 24 h, IL-1β release (J) in the cell supernatant was measured with ELISA; endogenous NLRP1 (K) was detected with Western blot. 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, one-way ANOVA in (D, J), two-tailed unpaired Student’s t-test in (B, F, H).
Supplier Page from Sino Biological, Inc. for Human PYCARD transcript variant 1 Gene ORF cDNA clone expression plasmid, C-HA tag