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(A–C) BMDMs were infected with ASFV (MOI = 0.1/1/10) for 24 h. Cell viability (A) <t>,</t> <t>Casp-1</t> activity (B) , and LDH release ( C ) were measured using CCK-8 assay kit, Casp-1 activity assay kit, and LDH assay kit, respectively. (D) PAMs were infected with ASFV-GFP (MOI = 1) for 24 h, followed by propidium iodide (PI) staining to observe cell death. A positive control (LPS [60 ng/mL for 8 h] + nigericin [Nig, 2 μM for 2 h] ) was included. (E) BMDMs were infected with ASFV (MOI = 1) for 24 h. Expression of pyroptosis marker proteins (Casp-1, IL-1β, and GSDMD-N) was analyzed by WB. (F) PAMs were infected with ASFV at an MOI of 1 for 24 h, fixed with 2% glutaraldehyde, and processed for transmission electron microscopy (TEM). Arrows indicate plasma membrane rupture. ( G ) iPAM cells were infected with ASFV at an MOI of 1. Formation of ASC specks and membrane localization of N-GSDMD were observed by immunofluorescence assay (IFA). ( H ) iPAM cells were infected with ASFV (MOI = 1) or positive control (LPS + Nig). ASC oligomerization was detected by WB using disuccinimidyl suberate (DSS) crosslinking. (I) PBMCs were infected with ASFV (MOI = 1) for 24 h. Expression of pyroptosis markers (Casp-1, IL-1β, N-GSDMD) and phosphorylation of IκBα and p65 in the NF-κB pathway were analyzed by WB. (J) PBMCs were infected with ASFV (MOI = 1) for 24 h, followed by PI staining to observe cell death. (K) PBMCs were infected with ASFV (MOI = 1) for 24 hours. Cell viability, Casp-1 activity, IL-1β transcription, and ASFV p72 mRNA level were assessed by CCK-8 assay, Casp-1 activity kit, qPCR, and qPCR, respectively. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001; statistical significance was determined by one-way ANOVA.

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1) Product Images from "African swine fever virus pEP364R acts as an important inflammatory-inducing factor to activate NLRP3 inflammasome-mediated pyroptosis by regulating DDX3X"

Article Title: African swine fever virus pEP364R acts as an important inflammatory-inducing factor to activate NLRP3 inflammasome-mediated pyroptosis by regulating DDX3X

Journal: PLOS Pathogens

doi: 10.1371/journal.ppat.1013874

(A–C) BMDMs were infected with ASFV (MOI = 0.1/1/10) for 24 h. Cell viability (A) , Casp-1 activity (B) , and LDH release ( C ) were measured using CCK-8 assay kit, Casp-1 activity assay kit, and LDH assay kit, respectively. (D) PAMs were infected with ASFV-GFP (MOI = 1) for 24 h, followed by propidium iodide (PI) staining to observe cell death. A positive control (LPS [60 ng/mL for 8 h] + nigericin [Nig, 2 μM for 2 h] ) was included. (E) BMDMs were infected with ASFV (MOI = 1) for 24 h. Expression of pyroptosis marker proteins (Casp-1, IL-1β, and GSDMD-N) was analyzed by WB. (F) PAMs were infected with ASFV at an MOI of 1 for 24 h, fixed with 2% glutaraldehyde, and processed for transmission electron microscopy (TEM). Arrows indicate plasma membrane rupture. ( G ) iPAM cells were infected with ASFV at an MOI of 1. Formation of ASC specks and membrane localization of N-GSDMD were observed by immunofluorescence assay (IFA). ( H ) iPAM cells were infected with ASFV (MOI = 1) or positive control (LPS + Nig). ASC oligomerization was detected by WB using disuccinimidyl suberate (DSS) crosslinking. (I) PBMCs were infected with ASFV (MOI = 1) for 24 h. Expression of pyroptosis markers (Casp-1, IL-1β, N-GSDMD) and phosphorylation of IκBα and p65 in the NF-κB pathway were analyzed by WB. (J) PBMCs were infected with ASFV (MOI = 1) for 24 h, followed by PI staining to observe cell death. (K) PBMCs were infected with ASFV (MOI = 1) for 24 hours. Cell viability, Casp-1 activity, IL-1β transcription, and ASFV p72 mRNA level were assessed by CCK-8 assay, Casp-1 activity kit, qPCR, and qPCR, respectively. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001; statistical significance was determined by one-way ANOVA.

Figure Legend Snippet: (A–C) BMDMs were infected with ASFV (MOI = 0.1/1/10) for 24 h. Cell viability (A) , Casp-1 activity (B) , and LDH release ( C ) were measured using CCK-8 assay kit, Casp-1 activity assay kit, and LDH assay kit, respectively. (D) PAMs were infected with ASFV-GFP (MOI = 1) for 24 h, followed by propidium iodide (PI) staining to observe cell death. A positive control (LPS [60 ng/mL for 8 h] + nigericin [Nig, 2 μM for 2 h] ) was included. (E) BMDMs were infected with ASFV (MOI = 1) for 24 h. Expression of pyroptosis marker proteins (Casp-1, IL-1β, and GSDMD-N) was analyzed by WB. (F) PAMs were infected with ASFV at an MOI of 1 for 24 h, fixed with 2% glutaraldehyde, and processed for transmission electron microscopy (TEM). Arrows indicate plasma membrane rupture. ( G ) iPAM cells were infected with ASFV at an MOI of 1. Formation of ASC specks and membrane localization of N-GSDMD were observed by immunofluorescence assay (IFA). ( H ) iPAM cells were infected with ASFV (MOI = 1) or positive control (LPS + Nig). ASC oligomerization was detected by WB using disuccinimidyl suberate (DSS) crosslinking. (I) PBMCs were infected with ASFV (MOI = 1) for 24 h. Expression of pyroptosis markers (Casp-1, IL-1β, N-GSDMD) and phosphorylation of IκBα and p65 in the NF-κB pathway were analyzed by WB. (J) PBMCs were infected with ASFV (MOI = 1) for 24 h, followed by PI staining to observe cell death. (K) PBMCs were infected with ASFV (MOI = 1) for 24 hours. Cell viability, Casp-1 activity, IL-1β transcription, and ASFV p72 mRNA level were assessed by CCK-8 assay, Casp-1 activity kit, qPCR, and qPCR, respectively. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001; statistical significance was determined by one-way ANOVA.

Techniques Used: Infection, Activity Assay, CCK-8 Assay, Lactate Dehydrogenase Assay, Staining, Positive Control, Expressing, Marker, Transmission Assay, Electron Microscopy, Clinical Proteomics, Membrane, Immunofluorescence, Phospho-proteomics

(A and B) PAMs were pretreated with the Casp-1 inhibitor VX765 (10 μM, 2 h) ( A ) or the NLRP3 inhibitor MCC950 (10 μM, 1 h) (B) , followed by ASFV infection (MOI = 1) for 24 h. DMSO was used as a control. IL-1β secretion was measured by ELISA, and pyroptosis biomarkers (Casp-1, IL-1β, N-GSDMD) expression were determined by WB. (C) PAMs were pretreated with VX765 or MCC950 as in (A and B) , infected with ASFV (MOI = 1) for 24 h, and subjected to PI staining. ASFV-induced cell death was observed. (D–F) PAMs were transfected with shASC (D) , shCasp-1 (E) , or shNLRP3 ( F ) for 72 h, followed by ASFV infection (MOI = 1) for 24 h. shNC was used as a negative control. ELISA used for IL-1β secretion detection, and pyroptosis biomarkers expression were determined by WB. (G) PAMs were transfected with shASC, shCasp-1, or shNLRP3 for 72 h, infected with ASFV (MOI = 1) for 24 h, and subjected to PI staining. LPS + Nig served as a positive control. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001; statistical significance was determined by one-way ANOVA.

Figure Legend Snippet: (A and B) PAMs were pretreated with the Casp-1 inhibitor VX765 (10 μM, 2 h) ( A ) or the NLRP3 inhibitor MCC950 (10 μM, 1 h) (B) , followed by ASFV infection (MOI = 1) for 24 h. DMSO was used as a control. IL-1β secretion was measured by ELISA, and pyroptosis biomarkers (Casp-1, IL-1β, N-GSDMD) expression were determined by WB. (C) PAMs were pretreated with VX765 or MCC950 as in (A and B) , infected with ASFV (MOI = 1) for 24 h, and subjected to PI staining. ASFV-induced cell death was observed. (D–F) PAMs were transfected with shASC (D) , shCasp-1 (E) , or shNLRP3 ( F ) for 72 h, followed by ASFV infection (MOI = 1) for 24 h. shNC was used as a negative control. ELISA used for IL-1β secretion detection, and pyroptosis biomarkers expression were determined by WB. (G) PAMs were transfected with shASC, shCasp-1, or shNLRP3 for 72 h, infected with ASFV (MOI = 1) for 24 h, and subjected to PI staining. LPS + Nig served as a positive control. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001; statistical significance was determined by one-way ANOVA.

Techniques Used: Infection, Control, Enzyme-linked Immunosorbent Assay, Expressing, Staining, Transfection, Negative Control, Positive Control

(A) PK-15 cells, with activated NLRP3 inflammasome components, were transfected with 2 μg of each of the 185 plasmids encoding ASFV proteins. IL-1β secretion was screened by ELISA to identify proteins inducing high IL-1β levels. ( B ) iPAMs cells were transfected with 2 μg of EP364R plasmid. At 24 h post-transfection (h.p.t), cells were fixed and the subcellular localization of EP364R was observed by confocal microscopy. Scale bars, 20 μm. (C) BMDMs cells were transfected with 2 μg of EP364R plasmid. IL-1β secretion in the supernatant was measured by ELISA at 24 h. ( D ) iPAMs were transfected with increasing concentrations (0.25, 0.5, 1.0 μg) of EP364R plasmid, with 1 μg of empty vector (EV) plasmid as a control. At 24 h, 10 μl of CCK-8 reagent was added, followed by incubation at 37°C for 1 hour. Absorbance was measured at 450 nm. ( E ) iPAMs were transfected with 2 μg of EP364R plasmid. Lactate dehydrogenase (LDH) release was detected using an LDH assay kit at 0, 12, and 24 h. ( F ) iPAMs were transfected with 2 μg of EP364R plasmid, alongside blank control, positive control [LPS (60 ng/mL for 8 h) + Nigericin (Nig, 2 μM for 2 h) ], liposome (4 μl) control, and negative control (EV, 2 μg). At 24 h, cells were stained with propidium iodide (PI) and cell death was observed by fluorescence microscopy. (G) Cell morphology was observed by microscopy 24 h after transfection of iPAMs with 2 μg of EP364R plasmid. ( H ) iPAMs were co-transfected with plasmids for NLRP3 (3 μg), pro-IL-1β (3 μg), ASC (1 μg), pro-Casp-1 (1 μg), and increasing amounts of EP364R (0, 2, 4, 6 μg). Cells were harvested and lysed 24 h p.t., and IL-1β secretion was analyzed by WB. ( I ) iPAMs were transfected with 2 μg of EP364R plasmid, with LPS + Nig treatment as a positive control. Cells were lysed 24 h p.t., and the expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB. ( J ) iPAMs were transfected with 2 μg of EP364R plasmid, with LPS + Nig as a positive control. At 24 h p.t., cell lysates were centrifuged. The supernatant was prepared as the ‘input’ sample. The cell pellet was resuspended in 100 μl PBS, cross-linked with 2 mM DSS at 37°C for 30 min, and then directly mixed with protein loading buffer to prepare the ‘pellet’ sample. ASC and GSDMD-N oligomerization were detected by WB. ( K ) iPAMs were transfected with 2 μg of EP364R plasmid. At 24 h p.t., cells were fixed, and ASC speck formation and GSDMD-N localization were observed by confocal microscopy. Scale bars, 20 μm. A P value less than 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure Legend Snippet: (A) PK-15 cells, with activated NLRP3 inflammasome components, were transfected with 2 μg of each of the 185 plasmids encoding ASFV proteins. IL-1β secretion was screened by ELISA to identify proteins inducing high IL-1β levels. ( B ) iPAMs cells were transfected with 2 μg of EP364R plasmid. At 24 h post-transfection (h.p.t), cells were fixed and the subcellular localization of EP364R was observed by confocal microscopy. Scale bars, 20 μm. (C) BMDMs cells were transfected with 2 μg of EP364R plasmid. IL-1β secretion in the supernatant was measured by ELISA at 24 h. ( D ) iPAMs were transfected with increasing concentrations (0.25, 0.5, 1.0 μg) of EP364R plasmid, with 1 μg of empty vector (EV) plasmid as a control. At 24 h, 10 μl of CCK-8 reagent was added, followed by incubation at 37°C for 1 hour. Absorbance was measured at 450 nm. ( E ) iPAMs were transfected with 2 μg of EP364R plasmid. Lactate dehydrogenase (LDH) release was detected using an LDH assay kit at 0, 12, and 24 h. ( F ) iPAMs were transfected with 2 μg of EP364R plasmid, alongside blank control, positive control [LPS (60 ng/mL for 8 h) + Nigericin (Nig, 2 μM for 2 h) ], liposome (4 μl) control, and negative control (EV, 2 μg). At 24 h, cells were stained with propidium iodide (PI) and cell death was observed by fluorescence microscopy. (G) Cell morphology was observed by microscopy 24 h after transfection of iPAMs with 2 μg of EP364R plasmid. ( H ) iPAMs were co-transfected with plasmids for NLRP3 (3 μg), pro-IL-1β (3 μg), ASC (1 μg), pro-Casp-1 (1 μg), and increasing amounts of EP364R (0, 2, 4, 6 μg). Cells were harvested and lysed 24 h p.t., and IL-1β secretion was analyzed by WB. ( I ) iPAMs were transfected with 2 μg of EP364R plasmid, with LPS + Nig treatment as a positive control. Cells were lysed 24 h p.t., and the expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB. ( J ) iPAMs were transfected with 2 μg of EP364R plasmid, with LPS + Nig as a positive control. At 24 h p.t., cell lysates were centrifuged. The supernatant was prepared as the ‘input’ sample. The cell pellet was resuspended in 100 μl PBS, cross-linked with 2 mM DSS at 37°C for 30 min, and then directly mixed with protein loading buffer to prepare the ‘pellet’ sample. ASC and GSDMD-N oligomerization were detected by WB. ( K ) iPAMs were transfected with 2 μg of EP364R plasmid. At 24 h p.t., cells were fixed, and ASC speck formation and GSDMD-N localization were observed by confocal microscopy. Scale bars, 20 μm. A P value less than 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques Used: Transfection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Confocal Microscopy, Control, CCK-8 Assay, Incubation, Lactate Dehydrogenase Assay, Positive Control, Negative Control, Staining, Fluorescence, Microscopy, Expressing

( A ) The pHBLV-EP364R lentiviral overexpression plasmid (10 μg) was co-transfected with lentiviral packaging helper plasmids psPAX2 (10 μg) and pMD2.G (10 μg) into HEK293T cells. Supernatants containing lentivirus were collected at 48 and 72 h p.t., and purified, and used to infect iPAMs. Stable puromycin-resistant cells expressing green fluorescent protein (GFP) were selected. Stable EP364R expression was confirmed by WB and PCR. ( B ) Casp-1, IL-1β, and GSDMD-N in EP364R-overexpressed iPAMs (LV-EP364R) were detected by WB, iPAMs expressing only GFP and resistance genes served as negative controls (LV-Vector), and Mock cells were normal iPAMs. ( C-D ) Transcriptional and secretion levels of cytokines IL-1β, IL-6, TNF-α, CXCL10, and IFN-β were measured by qPCR and ELISA in EP364R-overexpressed iPAMs. ( E ) Successful EP364R overexpressed lentivirus was purified and determined by WB at 72 h p.t. ( F ) Mice were intravenously injected twice with 5 × 10 7 TU of purified EP364R overexpression lentivirus, with a 48-h interval. Mice were randomly selected from each group and euthanized, and pathological changes in the heart, liver, spleen, lung, and kidney were examined. ( G ) Mice from the LV-EP364R and LV-Vector groups were randomly selected and euthanized. Tissues (heart, liver, spleen, lung, kidney; 1 g each) were harvested, homogenized, and proteins were extracted using tissue lysis buffer. Expression of IL-1β was detected by WB. And p value less than 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure Legend Snippet: ( A ) The pHBLV-EP364R lentiviral overexpression plasmid (10 μg) was co-transfected with lentiviral packaging helper plasmids psPAX2 (10 μg) and pMD2.G (10 μg) into HEK293T cells. Supernatants containing lentivirus were collected at 48 and 72 h p.t., and purified, and used to infect iPAMs. Stable puromycin-resistant cells expressing green fluorescent protein (GFP) were selected. Stable EP364R expression was confirmed by WB and PCR. ( B ) Casp-1, IL-1β, and GSDMD-N in EP364R-overexpressed iPAMs (LV-EP364R) were detected by WB, iPAMs expressing only GFP and resistance genes served as negative controls (LV-Vector), and Mock cells were normal iPAMs. ( C-D ) Transcriptional and secretion levels of cytokines IL-1β, IL-6, TNF-α, CXCL10, and IFN-β were measured by qPCR and ELISA in EP364R-overexpressed iPAMs. ( E ) Successful EP364R overexpressed lentivirus was purified and determined by WB at 72 h p.t. ( F ) Mice were intravenously injected twice with 5 × 10 7 TU of purified EP364R overexpression lentivirus, with a 48-h interval. Mice were randomly selected from each group and euthanized, and pathological changes in the heart, liver, spleen, lung, and kidney were examined. ( G ) Mice from the LV-EP364R and LV-Vector groups were randomly selected and euthanized. Tissues (heart, liver, spleen, lung, kidney; 1 g each) were harvested, homogenized, and proteins were extracted using tissue lysis buffer. Expression of IL-1β was detected by WB. And p value less than 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques Used: Over Expression, Plasmid Preparation, Transfection, Purification, Expressing, Enzyme-linked Immunosorbent Assay, Injection, Lysis

( A ) iPAMs were transfected with 2 μg of small hairpin RNA (shEP364R-1, -2, or -3) plasmids. At 72 h p.t., cells were transfected with 2 μg of EP364R plasmid for 24 h. small hairpin RNA Nonspecific shRNA (shNC) as a negative control. RNA was extracted for qPCR, and cells were lysed for WB analysis to screen for the most effective shEP364R. ( B ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV (MOI = 1). EP364R transcription levels were validated by qPCR 24 h p.i (hours post infection). ( C ) iPAMs were transfected with 50 nM of the selected siEP364R. At 72 hours post-transfection, cells were infected with ASFV at an MOI of 1. The culture supernatants were collected 24 hours post-infection, processed through repeated freeze-thaw cycles and lyophilization, and then analyzed by ELISA to quantify secreted IL-1β. ( D ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV (MOI = 1) for 24 h. IL-1β secretion was measured by ELISA, and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB. ( E ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. CCK-8 reagent (10 μl) was added, followed by incubation at 37°C for 1 h, and absorbance was measured at 450 nm. LPS and Nigericin stimulation group as a positive control. ( F ) iPAMs were transfected with 2 μg of shEP364R-3 or shNC plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. Intracellular Casp-1 activity was measured using a Caspase-1 activity assay kit. ( G ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. LDH release was detected using an LDH assay kit. ( H ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. Cells were stained with PI (10 μl) and cell death was observed by fluorescence microscopy. ( I ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. Cell lysates were centrifuged; the supernatant was prepared as the ‘input’ sample. The pellet was resuspended in PBS, cross-linked with DSS, incubated at 37°C for 30 min, and mixed with loading buffer to prepare the ‘pellet’ sample. ASC oligomerization was detected by WB. ( J ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. RNA was extracted, and transcriptional levels of IL-1β, IL-6, TNF-α, CXCL10, and EP364R were measured by qPCR. A P value less than 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure Legend Snippet: ( A ) iPAMs were transfected with 2 μg of small hairpin RNA (shEP364R-1, -2, or -3) plasmids. At 72 h p.t., cells were transfected with 2 μg of EP364R plasmid for 24 h. small hairpin RNA Nonspecific shRNA (shNC) as a negative control. RNA was extracted for qPCR, and cells were lysed for WB analysis to screen for the most effective shEP364R. ( B ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV (MOI = 1). EP364R transcription levels were validated by qPCR 24 h p.i (hours post infection). ( C ) iPAMs were transfected with 50 nM of the selected siEP364R. At 72 hours post-transfection, cells were infected with ASFV at an MOI of 1. The culture supernatants were collected 24 hours post-infection, processed through repeated freeze-thaw cycles and lyophilization, and then analyzed by ELISA to quantify secreted IL-1β. ( D ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV (MOI = 1) for 24 h. IL-1β secretion was measured by ELISA, and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB. ( E ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. CCK-8 reagent (10 μl) was added, followed by incubation at 37°C for 1 h, and absorbance was measured at 450 nm. LPS and Nigericin stimulation group as a positive control. ( F ) iPAMs were transfected with 2 μg of shEP364R-3 or shNC plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. Intracellular Casp-1 activity was measured using a Caspase-1 activity assay kit. ( G ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. LDH release was detected using an LDH assay kit. ( H ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. Cells were stained with PI (10 μl) and cell death was observed by fluorescence microscopy. ( I ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. Cell lysates were centrifuged; the supernatant was prepared as the ‘input’ sample. The pellet was resuspended in PBS, cross-linked with DSS, incubated at 37°C for 30 min, and mixed with loading buffer to prepare the ‘pellet’ sample. ASC oligomerization was detected by WB. ( J ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. RNA was extracted, and transcriptional levels of IL-1β, IL-6, TNF-α, CXCL10, and EP364R were measured by qPCR. A P value less than 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques Used: Transfection, Plasmid Preparation, shRNA, Negative Control, Infection, Lyophilization, Enzyme-linked Immunosorbent Assay, Expressing, CCK-8 Assay, Incubation, Positive Control, Activity Assay, Lactate Dehydrogenase Assay, Staining, Fluorescence, Microscopy

( A ) iPAMs were pretreated with the NLRP3 inhibitor MCC950 (10 μM) for 1 h, then transfected with 3 μg of EP364R plasmid. IL-1β secretion was measured by ELISA and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB 24 h p.t. ( B ) iPAMs were pretreated with the Casp-1 inhibitor VX765 (10 μM) for 2 h, then transfected with 3 μg of EP364R plasmid. IL-1β secretion was measured by ELISA and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB 24 h p.t. ( C-E ) iPAMs were transfected with 3 μg of shNLRP3 (C) , shASC (D) , or shCasp-1 ( E ) plasmids. At 72 h p.t., cells were transfected with 3 μg of EP364R plasmid. IL-1β secretion was measured by ELISA and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB 24 h later. ( F ) iPAMs were transfected with 3 μg of shNLRP3, shASC, or shCasp-1 plasmids. At 72 h p.t., cells were transfected with 3 μg of EP364R plasmid. At 24 h p.t., cells were stained with PI (10 μl) and cell death was observed by microscopy. (G) Bone marrow cells were isolated from NLRP3-/- knockout mice and differentiated for 7 days in medium containing L929 cell-conditioned supernatant. Cells were then transfected with 3 μg of EP364R plasmid. Transcriptional levels of EP364R and NLRP3 were measured by qPCR 24 h p.t., showing no significant difference in EP364R transcription but confirmed absence of NLRP3. (H) Bone marrow-derived macrophages (BMDMs) from NLRP3-/- mice (prepared as in G) were transfected with 3 μg of EP364R plasmid. IL-1β secretion was measured by ELISA and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB 24 h p.t. A P value less than 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure Legend Snippet: ( A ) iPAMs were pretreated with the NLRP3 inhibitor MCC950 (10 μM) for 1 h, then transfected with 3 μg of EP364R plasmid. IL-1β secretion was measured by ELISA and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB 24 h p.t. ( B ) iPAMs were pretreated with the Casp-1 inhibitor VX765 (10 μM) for 2 h, then transfected with 3 μg of EP364R plasmid. IL-1β secretion was measured by ELISA and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB 24 h p.t. ( C-E ) iPAMs were transfected with 3 μg of shNLRP3 (C) , shASC (D) , or shCasp-1 ( E ) plasmids. At 72 h p.t., cells were transfected with 3 μg of EP364R plasmid. IL-1β secretion was measured by ELISA and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB 24 h later. ( F ) iPAMs were transfected with 3 μg of shNLRP3, shASC, or shCasp-1 plasmids. At 72 h p.t., cells were transfected with 3 μg of EP364R plasmid. At 24 h p.t., cells were stained with PI (10 μl) and cell death was observed by microscopy. (G) Bone marrow cells were isolated from NLRP3-/- knockout mice and differentiated for 7 days in medium containing L929 cell-conditioned supernatant. Cells were then transfected with 3 μg of EP364R plasmid. Transcriptional levels of EP364R and NLRP3 were measured by qPCR 24 h p.t., showing no significant difference in EP364R transcription but confirmed absence of NLRP3. (H) Bone marrow-derived macrophages (BMDMs) from NLRP3-/- mice (prepared as in G) were transfected with 3 μg of EP364R plasmid. IL-1β secretion was measured by ELISA and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB 24 h p.t. A P value less than 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques Used: Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Expressing, Staining, Microscopy, Isolation, Knock-Out, Derivative Assay

Upon infection of domestic swine tissues, the ASFV-encoded protein EP364R binds to a specific spatial structure of DDX3X, which triggers the oligomerization of the NLRP3 inflammasome. This activation leads to the cleavage and activation of Casp-1, which in turn executes pyroptotic cell death and facilitates the release of pro‑inflammatory cytokines and damage‑associated molecular patterns (DAMPs). This cascade is likely a key driver of the cytokine storm‑mediate tissues damage during infection. HAMNO, a small molecule targeting EP364R, inhibits ASFV replication through disruption of the EP364R-DDX3X interaction.

Figure Legend Snippet: Upon infection of domestic swine tissues, the ASFV-encoded protein EP364R binds to a specific spatial structure of DDX3X, which triggers the oligomerization of the NLRP3 inflammasome. This activation leads to the cleavage and activation of Casp-1, which in turn executes pyroptotic cell death and facilitates the release of pro‑inflammatory cytokines and damage‑associated molecular patterns (DAMPs). This cascade is likely a key driver of the cytokine storm‑mediate tissues damage during infection. HAMNO, a small molecule targeting EP364R, inhibits ASFV replication through disruption of the EP364R-DDX3X interaction.

Techniques Used: Infection, Activation Assay, Disruption

Infection:

Article Title: African swine fever virus pEP364R acts as an important inflammatory-inducing factor to activate NLRP3 inflammasome-mediated pyroptosis by regulating DDX3X
Article Snippet: Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (H + L) and HRP-labeled goat anti-mouse IgG (H + L) were purchased from ZSGB-BIO.

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Infection:

Activity Assay:

CCK-8 Assay:

Lactate Dehydrogenase Assay:

Staining:

Positive Control:

Expressing:

Marker:

Transmission Assay:

Electron Microscopy:

Clinical Proteomics:

Membrane:

Immunofluorescence:

Phospho-proteomics:

Control:

Enzyme-linked Immunosorbent Assay:

Transfection:

Negative Control:

Plasmid Preparation:

Confocal Microscopy:

Incubation:

Fluorescence:

Microscopy:

Over Expression:

Purification:

Injection:

Lysis:

shRNA:

Lyophilization:

Isolation:

Knock-Out:

Derivative Assay:

Activation Assay:

Disruption:

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