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IL-1β and TNF-α production in THP-1 cells and inflammasome-reconstructed 293T cells. (A) THP-1 cells were infected with the parental strain Nc1 or the NcGRA6-, NcGRA7-, or NcGRA14-deficient (KO) parasites at a multiplicity of infection (MOI) of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. (B) 293T cells were transfected with inflammasome-reconstruction plasmids encoding <t>NLRP3,</t> ASC, procaspase-1, and pro-IL-1β, together with NcGRA7 cDNA or an empty vector. Untransfected 293T cells were used as a negative control (no plasmid). At 20 h posttransfection, the culture supernatants were collected for analysis. (C–H) THP-1 cells were pretreated with 10 μM MCC950 (an NLARP3 inhibitor), 100 μM <t>VX765</t> (a CASP1 inhibitor), and 18 μM SN50 (an NF-κB inhibitor) for 2 hr and then infected with the Nc1 strain of N. caninum at a MOI of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. Each value represents the mean ± SD of 4 replicates (technical replicates) in one representative experiment. Each experiment (biological replicate) was repeated two (G, H) , three (A, D, E, F) and four times (B, C) . Statistically significant differences according to one-way ANOVA or two-way ANOVA and a Tukey–Kramer post hoc analysis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).
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IL-1β and TNF-α production in THP-1 cells and inflammasome-reconstructed 293T cells. (A) THP-1 cells were infected with the parental strain Nc1 or the NcGRA6-, NcGRA7-, or NcGRA14-deficient (KO) parasites at a multiplicity of infection (MOI) of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. (B) 293T cells were transfected with inflammasome-reconstruction plasmids encoding <t>NLRP3,</t> ASC, procaspase-1, and pro-IL-1β, together with NcGRA7 cDNA or an empty vector. Untransfected 293T cells were used as a negative control (no plasmid). At 20 h posttransfection, the culture supernatants were collected for analysis. (C–H) THP-1 cells were pretreated with 10 μM MCC950 (an NLARP3 inhibitor), 100 μM <t>VX765</t> (a CASP1 inhibitor), and 18 μM SN50 (an NF-κB inhibitor) for 2 hr and then infected with the Nc1 strain of N. caninum at a MOI of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. Each value represents the mean ± SD of 4 replicates (technical replicates) in one representative experiment. Each experiment (biological replicate) was repeated two (G, H) , three (A, D, E, F) and four times (B, C) . Statistically significant differences according to one-way ANOVA or two-way ANOVA and a Tukey–Kramer post hoc analysis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).
G810548 Genistin Fam Genebio Technology N A Vx765 Selleck, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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IL-1β and TNF-α production in THP-1 cells and inflammasome-reconstructed 293T cells. (A) THP-1 cells were infected with the parental strain Nc1 or the NcGRA6-, NcGRA7-, or NcGRA14-deficient (KO) parasites at a multiplicity of infection (MOI) of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. (B) 293T cells were transfected with inflammasome-reconstruction plasmids encoding <t>NLRP3,</t> ASC, procaspase-1, and pro-IL-1β, together with NcGRA7 cDNA or an empty vector. Untransfected 293T cells were used as a negative control (no plasmid). At 20 h posttransfection, the culture supernatants were collected for analysis. (C–H) THP-1 cells were pretreated with 10 μM MCC950 (an NLARP3 inhibitor), 100 μM <t>VX765</t> (a CASP1 inhibitor), and 18 μM SN50 (an NF-κB inhibitor) for 2 hr and then infected with the Nc1 strain of N. caninum at a MOI of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. Each value represents the mean ± SD of 4 replicates (technical replicates) in one representative experiment. Each experiment (biological replicate) was repeated two (G, H) , three (A, D, E, F) and four times (B, C) . Statistically significant differences according to one-way ANOVA or two-way ANOVA and a Tukey–Kramer post hoc analysis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).
Vx765, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cell pyroptosis inhibitor vx765  (MedChemExpress)
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IL-1β and TNF-α production in THP-1 cells and inflammasome-reconstructed 293T cells. (A) THP-1 cells were infected with the parental strain Nc1 or the NcGRA6-, NcGRA7-, or NcGRA14-deficient (KO) parasites at a multiplicity of infection (MOI) of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. (B) 293T cells were transfected with inflammasome-reconstruction plasmids encoding <t>NLRP3,</t> ASC, procaspase-1, and pro-IL-1β, together with NcGRA7 cDNA or an empty vector. Untransfected 293T cells were used as a negative control (no plasmid). At 20 h posttransfection, the culture supernatants were collected for analysis. (C–H) THP-1 cells were pretreated with 10 μM MCC950 (an NLARP3 inhibitor), 100 μM <t>VX765</t> (a CASP1 inhibitor), and 18 μM SN50 (an NF-κB inhibitor) for 2 hr and then infected with the Nc1 strain of N. caninum at a MOI of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. Each value represents the mean ± SD of 4 replicates (technical replicates) in one representative experiment. Each experiment (biological replicate) was repeated two (G, H) , three (A, D, E, F) and four times (B, C) . Statistically significant differences according to one-way ANOVA or two-way ANOVA and a Tukey–Kramer post hoc analysis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).
Cell Pyroptosis Inhibitor Vx765, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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IL-1β and TNF-α production in THP-1 cells and inflammasome-reconstructed 293T cells. (A) THP-1 cells were infected with the parental strain Nc1 or the NcGRA6-, NcGRA7-, or NcGRA14-deficient (KO) parasites at a multiplicity of infection (MOI) of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. (B) 293T cells were transfected with inflammasome-reconstruction plasmids encoding NLRP3, ASC, procaspase-1, and pro-IL-1β, together with NcGRA7 cDNA or an empty vector. Untransfected 293T cells were used as a negative control (no plasmid). At 20 h posttransfection, the culture supernatants were collected for analysis. (C–H) THP-1 cells were pretreated with 10 μM MCC950 (an NLARP3 inhibitor), 100 μM VX765 (a CASP1 inhibitor), and 18 μM SN50 (an NF-κB inhibitor) for 2 hr and then infected with the Nc1 strain of N. caninum at a MOI of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. Each value represents the mean ± SD of 4 replicates (technical replicates) in one representative experiment. Each experiment (biological replicate) was repeated two (G, H) , three (A, D, E, F) and four times (B, C) . Statistically significant differences according to one-way ANOVA or two-way ANOVA and a Tukey–Kramer post hoc analysis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Journal: Frontiers in Immunology

Article Title: Mitochondrial damage and IL-1β production in monocytes caused by Neospora caninum infection are mediated by dense granule protein 7 and prohibitins

doi: 10.3389/fimmu.2025.1408992

Figure Lengend Snippet: IL-1β and TNF-α production in THP-1 cells and inflammasome-reconstructed 293T cells. (A) THP-1 cells were infected with the parental strain Nc1 or the NcGRA6-, NcGRA7-, or NcGRA14-deficient (KO) parasites at a multiplicity of infection (MOI) of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. (B) 293T cells were transfected with inflammasome-reconstruction plasmids encoding NLRP3, ASC, procaspase-1, and pro-IL-1β, together with NcGRA7 cDNA or an empty vector. Untransfected 293T cells were used as a negative control (no plasmid). At 20 h posttransfection, the culture supernatants were collected for analysis. (C–H) THP-1 cells were pretreated with 10 μM MCC950 (an NLARP3 inhibitor), 100 μM VX765 (a CASP1 inhibitor), and 18 μM SN50 (an NF-κB inhibitor) for 2 hr and then infected with the Nc1 strain of N. caninum at a MOI of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. Each value represents the mean ± SD of 4 replicates (technical replicates) in one representative experiment. Each experiment (biological replicate) was repeated two (G, H) , three (A, D, E, F) and four times (B, C) . Statistically significant differences according to one-way ANOVA or two-way ANOVA and a Tukey–Kramer post hoc analysis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: MCC950 (an inhibitor of NLRP3 inflammasome activation by the inhibition of IL-1β release; Cayman Chemical), VX765 (an inhibitor of NLRP3 inflammasome activation by the inhibition of caspase 1 and IL-1β cleavage and release; LKT Laboratories), SN50 (an inhibitor of the nuclear transcription factor κB; Selleck, Houston, TX, USA), a carbonyl cyanide m-chlorophenyl hydrazone (CCCP, a uncoupling agent for oxidative phosphorylation that inhibits mitochondrial function; FUJFILM Wako Pure Chemical Corporation), rocaglamid A (Cayman, Ann Arbor, MI, USA) with biochemical research grades were used in this study.

Techniques: Infection, Transfection, Plasmid Preparation, Negative Control