mce extem, rotem® extrinsically activated test with tf Search Results


recombinant ahcyl1  (OriGene)
94
OriGene recombinant ahcyl1
Recombinant Ahcyl1, supplied by OriGene, 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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Average 94 stars, based on 1 article reviews
recombinant ahcyl1 - by Bioz Stars, 2026-09
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erastin  (MedChemExpress)
99
MedChemExpress erastin
Erastin, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 1 article reviews
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2-[(4r)-4-[(1r,3as,3br,5ar,7r,9as,9bs,11s,11ar)-7,11-dihydroxy-9a,11a-dimethyl-hexadecahydro-1h-cyclopenta[a]phenanthren-1-yl]pentanamido]acetic acid  (Enamine Ltd)
94
Enamine Ltd 2-[(4r)-4-[(1r,3as,3br,5ar,7r,9as,9bs,11s,11ar)-7,11-dihydroxy-9a,11a-dimethyl-hexadecahydro-1h-cyclopenta[a]phenanthren-1-yl]pentanamido]acetic acid
2 [(4r) 4 [(1r,3as,3br,5ar,7r,9as,9bs,11s,11ar) 7,11 Dihydroxy 9a,11a Dimethyl Hexadecahydro 1h Cyclopenta[A]Phenanthren 1 Yl]Pentanamido]Acetic Acid, supplied by Enamine Ltd, 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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Average 94 stars, based on 1 article reviews
2-[(4r)-4-[(1r,3as,3br,5ar,7r,9as,9bs,11s,11ar)-7,11-dihydroxy-9a,11a-dimethyl-hexadecahydro-1h-cyclopenta[a]phenanthren-1-yl]pentanamido]acetic acid - by Bioz Stars, 2026-09
94/100 stars
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palytoxin  (FUJIFILM)
90
FUJIFILM palytoxin
A. Western blot of PhosTag SDS-PAGE (ZAKɑ and MK2) or SDS-PAGE (p-p38,p-JNK and HSP70) N/TERT cells treated with anisomycin (1µM), nigericin (5µg/mL), valinomycin (5µM), salinomycin (5µM), monensin (5µM), gramicidin (1µM), nonactin (5µM), BME-44 (5µM), lasalocid (5µM), <t>palytoxin</t> (100pM) or VbP (3µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments. B. Anti-puromycin immunoblots of N/TERT cells subjected to harringtonine run-off. Cells were treated for 3 hours with valinomycin (5µM), salinomycin (5µM), monensin (5µM), nigericin (5µg/mL) before harringtonine addition (2µg/mL) for the respective durations. Nascent peptides were then labeled with puromycin (10µg/mL) for 10 mins. Ponceau staining was used as the loading control. Immunoblot representative of three replicate experiments. C. Same as (B) except cells were treated with gramicidin (1µM), lasalocid (5µM) or palytoxin (100pM). D. ELISA showing IL-1β secretion in primary keratinocytes following overnight treatment with various potassium ionophores and anisomycin. E. Western blot analysis of primary keratinocytes showing GSDMD (full length and cleaved), pro-IL1β, cleaved IL1β and GAPDH as loading control upon overnight treatment with nigericin, valinomycin (10µM), salinomycin (10µM) or monensin (10µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments.
Palytoxin, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews
palytoxin - by Bioz Stars, 2026-09
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cpz  (Selleck Chemicals)
94
Selleck Chemicals cpz
A. Western blot of PhosTag SDS-PAGE (ZAKɑ and MK2) or SDS-PAGE (p-p38,p-JNK and HSP70) N/TERT cells treated with anisomycin (1µM), nigericin (5µg/mL), valinomycin (5µM), salinomycin (5µM), monensin (5µM), gramicidin (1µM), nonactin (5µM), BME-44 (5µM), lasalocid (5µM), <t>palytoxin</t> (100pM) or VbP (3µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments. B. Anti-puromycin immunoblots of N/TERT cells subjected to harringtonine run-off. Cells were treated for 3 hours with valinomycin (5µM), salinomycin (5µM), monensin (5µM), nigericin (5µg/mL) before harringtonine addition (2µg/mL) for the respective durations. Nascent peptides were then labeled with puromycin (10µg/mL) for 10 mins. Ponceau staining was used as the loading control. Immunoblot representative of three replicate experiments. C. Same as (B) except cells were treated with gramicidin (1µM), lasalocid (5µM) or palytoxin (100pM). D. ELISA showing IL-1β secretion in primary keratinocytes following overnight treatment with various potassium ionophores and anisomycin. E. Western blot analysis of primary keratinocytes showing GSDMD (full length and cleaved), pro-IL1β, cleaved IL1β and GAPDH as loading control upon overnight treatment with nigericin, valinomycin (10µM), salinomycin (10µM) or monensin (10µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments.
Cpz, supplied by Selleck Chemicals, 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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Average 94 stars, based on 1 article reviews
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u0126  (MedChemExpress)
98
MedChemExpress u0126
A. Western blot of PhosTag SDS-PAGE (ZAKɑ and MK2) or SDS-PAGE (p-p38,p-JNK and HSP70) N/TERT cells treated with anisomycin (1µM), nigericin (5µg/mL), valinomycin (5µM), salinomycin (5µM), monensin (5µM), gramicidin (1µM), nonactin (5µM), BME-44 (5µM), lasalocid (5µM), <t>palytoxin</t> (100pM) or VbP (3µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments. B. Anti-puromycin immunoblots of N/TERT cells subjected to harringtonine run-off. Cells were treated for 3 hours with valinomycin (5µM), salinomycin (5µM), monensin (5µM), nigericin (5µg/mL) before harringtonine addition (2µg/mL) for the respective durations. Nascent peptides were then labeled with puromycin (10µg/mL) for 10 mins. Ponceau staining was used as the loading control. Immunoblot representative of three replicate experiments. C. Same as (B) except cells were treated with gramicidin (1µM), lasalocid (5µM) or palytoxin (100pM). D. ELISA showing IL-1β secretion in primary keratinocytes following overnight treatment with various potassium ionophores and anisomycin. E. Western blot analysis of primary keratinocytes showing GSDMD (full length and cleaved), pro-IL1β, cleaved IL1β and GAPDH as loading control upon overnight treatment with nigericin, valinomycin (10µM), salinomycin (10µM) or monensin (10µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments.
U0126, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 98 stars, based on 1 article reviews
u0126 - by Bioz Stars, 2026-09
98/100 stars
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dehydrocholic acid  (Enamine Ltd)
94
Enamine Ltd dehydrocholic acid
A. Western blot of PhosTag SDS-PAGE (ZAKɑ and MK2) or SDS-PAGE (p-p38,p-JNK and HSP70) N/TERT cells treated with anisomycin (1µM), nigericin (5µg/mL), valinomycin (5µM), salinomycin (5µM), monensin (5µM), gramicidin (1µM), nonactin (5µM), BME-44 (5µM), lasalocid (5µM), <t>palytoxin</t> (100pM) or VbP (3µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments. B. Anti-puromycin immunoblots of N/TERT cells subjected to harringtonine run-off. Cells were treated for 3 hours with valinomycin (5µM), salinomycin (5µM), monensin (5µM), nigericin (5µg/mL) before harringtonine addition (2µg/mL) for the respective durations. Nascent peptides were then labeled with puromycin (10µg/mL) for 10 mins. Ponceau staining was used as the loading control. Immunoblot representative of three replicate experiments. C. Same as (B) except cells were treated with gramicidin (1µM), lasalocid (5µM) or palytoxin (100pM). D. ELISA showing IL-1β secretion in primary keratinocytes following overnight treatment with various potassium ionophores and anisomycin. E. Western blot analysis of primary keratinocytes showing GSDMD (full length and cleaved), pro-IL1β, cleaved IL1β and GAPDH as loading control upon overnight treatment with nigericin, valinomycin (10µM), salinomycin (10µM) or monensin (10µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments.
Dehydrocholic Acid, supplied by Enamine Ltd, 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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Average 94 stars, based on 1 article reviews
dehydrocholic acid - by Bioz Stars, 2026-09
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ursodeoxycholic acid  (Enamine Ltd)
99
Enamine Ltd ursodeoxycholic acid
A. Western blot of PhosTag SDS-PAGE (ZAKɑ and MK2) or SDS-PAGE (p-p38,p-JNK and HSP70) N/TERT cells treated with anisomycin (1µM), nigericin (5µg/mL), valinomycin (5µM), salinomycin (5µM), monensin (5µM), gramicidin (1µM), nonactin (5µM), BME-44 (5µM), lasalocid (5µM), <t>palytoxin</t> (100pM) or VbP (3µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments. B. Anti-puromycin immunoblots of N/TERT cells subjected to harringtonine run-off. Cells were treated for 3 hours with valinomycin (5µM), salinomycin (5µM), monensin (5µM), nigericin (5µg/mL) before harringtonine addition (2µg/mL) for the respective durations. Nascent peptides were then labeled with puromycin (10µg/mL) for 10 mins. Ponceau staining was used as the loading control. Immunoblot representative of three replicate experiments. C. Same as (B) except cells were treated with gramicidin (1µM), lasalocid (5µM) or palytoxin (100pM). D. ELISA showing IL-1β secretion in primary keratinocytes following overnight treatment with various potassium ionophores and anisomycin. E. Western blot analysis of primary keratinocytes showing GSDMD (full length and cleaved), pro-IL1β, cleaved IL1β and GAPDH as loading control upon overnight treatment with nigericin, valinomycin (10µM), salinomycin (10µM) or monensin (10µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments.
Ursodeoxycholic Acid, supplied by Enamine Ltd, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 1 article reviews
ursodeoxycholic acid - by Bioz Stars, 2026-09
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chenodeoxycholic acid  (Enamine Ltd)
99
Enamine Ltd chenodeoxycholic acid
A. Western blot of PhosTag SDS-PAGE (ZAKɑ and MK2) or SDS-PAGE (p-p38,p-JNK and HSP70) N/TERT cells treated with anisomycin (1µM), nigericin (5µg/mL), valinomycin (5µM), salinomycin (5µM), monensin (5µM), gramicidin (1µM), nonactin (5µM), BME-44 (5µM), lasalocid (5µM), <t>palytoxin</t> (100pM) or VbP (3µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments. B. Anti-puromycin immunoblots of N/TERT cells subjected to harringtonine run-off. Cells were treated for 3 hours with valinomycin (5µM), salinomycin (5µM), monensin (5µM), nigericin (5µg/mL) before harringtonine addition (2µg/mL) for the respective durations. Nascent peptides were then labeled with puromycin (10µg/mL) for 10 mins. Ponceau staining was used as the loading control. Immunoblot representative of three replicate experiments. C. Same as (B) except cells were treated with gramicidin (1µM), lasalocid (5µM) or palytoxin (100pM). D. ELISA showing IL-1β secretion in primary keratinocytes following overnight treatment with various potassium ionophores and anisomycin. E. Western blot analysis of primary keratinocytes showing GSDMD (full length and cleaved), pro-IL1β, cleaved IL1β and GAPDH as loading control upon overnight treatment with nigericin, valinomycin (10µM), salinomycin (10µM) or monensin (10µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments.
Chenodeoxycholic Acid, supplied by Enamine Ltd, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 1 article reviews
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celltiter-glo 3d cell viability assay  (Promega)
90
Promega celltiter-glo 3d cell viability assay
A. Western blot of PhosTag SDS-PAGE (ZAKɑ and MK2) or SDS-PAGE (p-p38,p-JNK and HSP70) N/TERT cells treated with anisomycin (1µM), nigericin (5µg/mL), valinomycin (5µM), salinomycin (5µM), monensin (5µM), gramicidin (1µM), nonactin (5µM), BME-44 (5µM), lasalocid (5µM), <t>palytoxin</t> (100pM) or VbP (3µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments. B. Anti-puromycin immunoblots of N/TERT cells subjected to harringtonine run-off. Cells were treated for 3 hours with valinomycin (5µM), salinomycin (5µM), monensin (5µM), nigericin (5µg/mL) before harringtonine addition (2µg/mL) for the respective durations. Nascent peptides were then labeled with puromycin (10µg/mL) for 10 mins. Ponceau staining was used as the loading control. Immunoblot representative of three replicate experiments. C. Same as (B) except cells were treated with gramicidin (1µM), lasalocid (5µM) or palytoxin (100pM). D. ELISA showing IL-1β secretion in primary keratinocytes following overnight treatment with various potassium ionophores and anisomycin. E. Western blot analysis of primary keratinocytes showing GSDMD (full length and cleaved), pro-IL1β, cleaved IL1β and GAPDH as loading control upon overnight treatment with nigericin, valinomycin (10µM), salinomycin (10µM) or monensin (10µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments.
Celltiter Glo 3d Cell Viability Assay, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews
celltiter-glo 3d cell viability assay - by Bioz Stars, 2026-09
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bafilomycin a1  (MedChemExpress)
99
MedChemExpress bafilomycin a1
A. Western blot of PhosTag SDS-PAGE (ZAKɑ and MK2) or SDS-PAGE (p-p38,p-JNK and HSP70) N/TERT cells treated with anisomycin (1µM), nigericin (5µg/mL), valinomycin (5µM), salinomycin (5µM), monensin (5µM), gramicidin (1µM), nonactin (5µM), BME-44 (5µM), lasalocid (5µM), <t>palytoxin</t> (100pM) or VbP (3µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments. B. Anti-puromycin immunoblots of N/TERT cells subjected to harringtonine run-off. Cells were treated for 3 hours with valinomycin (5µM), salinomycin (5µM), monensin (5µM), nigericin (5µg/mL) before harringtonine addition (2µg/mL) for the respective durations. Nascent peptides were then labeled with puromycin (10µg/mL) for 10 mins. Ponceau staining was used as the loading control. Immunoblot representative of three replicate experiments. C. Same as (B) except cells were treated with gramicidin (1µM), lasalocid (5µM) or palytoxin (100pM). D. ELISA showing IL-1β secretion in primary keratinocytes following overnight treatment with various potassium ionophores and anisomycin. E. Western blot analysis of primary keratinocytes showing GSDMD (full length and cleaved), pro-IL1β, cleaved IL1β and GAPDH as loading control upon overnight treatment with nigericin, valinomycin (10µM), salinomycin (10µM) or monensin (10µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments.
Bafilomycin A1, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rapamycin  (MedChemExpress)
99
MedChemExpress rapamycin
A. Western blot of PhosTag SDS-PAGE (ZAKɑ and MK2) or SDS-PAGE (p-p38,p-JNK and HSP70) N/TERT cells treated with anisomycin (1µM), nigericin (5µg/mL), valinomycin (5µM), salinomycin (5µM), monensin (5µM), gramicidin (1µM), nonactin (5µM), BME-44 (5µM), lasalocid (5µM), <t>palytoxin</t> (100pM) or VbP (3µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments. B. Anti-puromycin immunoblots of N/TERT cells subjected to harringtonine run-off. Cells were treated for 3 hours with valinomycin (5µM), salinomycin (5µM), monensin (5µM), nigericin (5µg/mL) before harringtonine addition (2µg/mL) for the respective durations. Nascent peptides were then labeled with puromycin (10µg/mL) for 10 mins. Ponceau staining was used as the loading control. Immunoblot representative of three replicate experiments. C. Same as (B) except cells were treated with gramicidin (1µM), lasalocid (5µM) or palytoxin (100pM). D. ELISA showing IL-1β secretion in primary keratinocytes following overnight treatment with various potassium ionophores and anisomycin. E. Western blot analysis of primary keratinocytes showing GSDMD (full length and cleaved), pro-IL1β, cleaved IL1β and GAPDH as loading control upon overnight treatment with nigericin, valinomycin (10µM), salinomycin (10µM) or monensin (10µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments.
Rapamycin, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


A. Western blot of PhosTag SDS-PAGE (ZAKɑ and MK2) or SDS-PAGE (p-p38,p-JNK and HSP70) N/TERT cells treated with anisomycin (1µM), nigericin (5µg/mL), valinomycin (5µM), salinomycin (5µM), monensin (5µM), gramicidin (1µM), nonactin (5µM), BME-44 (5µM), lasalocid (5µM), palytoxin (100pM) or VbP (3µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments. B. Anti-puromycin immunoblots of N/TERT cells subjected to harringtonine run-off. Cells were treated for 3 hours with valinomycin (5µM), salinomycin (5µM), monensin (5µM), nigericin (5µg/mL) before harringtonine addition (2µg/mL) for the respective durations. Nascent peptides were then labeled with puromycin (10µg/mL) for 10 mins. Ponceau staining was used as the loading control. Immunoblot representative of three replicate experiments. C. Same as (B) except cells were treated with gramicidin (1µM), lasalocid (5µM) or palytoxin (100pM). D. ELISA showing IL-1β secretion in primary keratinocytes following overnight treatment with various potassium ionophores and anisomycin. E. Western blot analysis of primary keratinocytes showing GSDMD (full length and cleaved), pro-IL1β, cleaved IL1β and GAPDH as loading control upon overnight treatment with nigericin, valinomycin (10µM), salinomycin (10µM) or monensin (10µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments.

Journal: bioRxiv

Article Title: Mechanistic basis for nigericin-induced NLRP1 inflammasome activation in human epithelial cells

doi: 10.1101/2023.06.23.546021

Figure Lengend Snippet: A. Western blot of PhosTag SDS-PAGE (ZAKɑ and MK2) or SDS-PAGE (p-p38,p-JNK and HSP70) N/TERT cells treated with anisomycin (1µM), nigericin (5µg/mL), valinomycin (5µM), salinomycin (5µM), monensin (5µM), gramicidin (1µM), nonactin (5µM), BME-44 (5µM), lasalocid (5µM), palytoxin (100pM) or VbP (3µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments. B. Anti-puromycin immunoblots of N/TERT cells subjected to harringtonine run-off. Cells were treated for 3 hours with valinomycin (5µM), salinomycin (5µM), monensin (5µM), nigericin (5µg/mL) before harringtonine addition (2µg/mL) for the respective durations. Nascent peptides were then labeled with puromycin (10µg/mL) for 10 mins. Ponceau staining was used as the loading control. Immunoblot representative of three replicate experiments. C. Same as (B) except cells were treated with gramicidin (1µM), lasalocid (5µM) or palytoxin (100pM). D. ELISA showing IL-1β secretion in primary keratinocytes following overnight treatment with various potassium ionophores and anisomycin. E. Western blot analysis of primary keratinocytes showing GSDMD (full length and cleaved), pro-IL1β, cleaved IL1β and GAPDH as loading control upon overnight treatment with nigericin, valinomycin (10µM), salinomycin (10µM) or monensin (10µM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments.

Article Snippet: The following drugs and chemicals were used as part of this study: Nigericin (Sigma, N7143), Anisomycin (ANS, MCE, #HY18982), Talabostat (VbP, MCE, #HY-13233), MCC950 (MCE, HY-12815A), ATP (Thermofisher Scientific, R0441), Belnacasan (MCE, HY-13205), Emricasan (MCE, HY-10396), Potassium chloride (Sigma Aldrich, P9541), BAPTA (MCE, HY-100168), Harringtonine (HTN, MCE, HY-N0862), Puromycin (PURO, Sigma, #P9620), M443 (MCE, HY-112274), Neflamapimod (p38i) (MCE, HY-10328), Bentamapimod (JNKi) (MCE, HY-14761), Valinomycin (MCE, HY-N6693), Salinomycin (MCE, HY-15597), Monensin (MCE, HY-N0150), Gramicidin (MCE, HY-P0163), Palytoxin (Fujifilm Wako, 165-26141), Nonactin (Sigma Aldrich, N2286), BME-44 (Sigma Aldrich 60397), Lasalocid (MCE, HY-B1071), Emetine (EME, MCE, HY-B1479B).

Techniques: Western Blot, SDS Page, Labeling, Staining, Enzyme-linked Immunosorbent Assay

A. Brightfield and PI inclusion images of primary keratinocytes upon nigericin (5µg/mL), lasalocid (5µM), gramicidin (1µM) or palytoxin (100pM) 0, 3 and 6 hours post treatment. Arrows indicate lysed cells that are PI negative. Representative data are shown for n = 3. B. Quantification of the percentage of PI positive Cas9 control and NLRP1 KO primary keratinocytes upon lasalocid (5µM) treatment. Cells were imaged at 15 minute intervals for 18 hours. C. Quantification of the percentage of PI positive primary keratinocytes with or without pre-treatment with M443 (1µM) before lasalocid treatment (5µM).Cells were imaged at 15 minute intervals for 18 hours. D. Quantification of the percentage of PI positive primary keratinocytes with or without pre-treatment with emricasan (5µM) before nigericin (5µg/mL) E. Quantification of the percentage of PI positive primary keratinocytes with or without pre-treatment with emricasan (5µM) before palytoxin (100pM) treatment.Cells were imaged at 15 minute intervals for 18 hours. F. Comparison of the inner diameters of the respective channels/pore to the diameters of H2O and ions. Diagram drawn to scale. G. Summary of how nigericin activates NLRP3 and NLRP1 in distinct human cell types.

Journal: bioRxiv

Article Title: Mechanistic basis for nigericin-induced NLRP1 inflammasome activation in human epithelial cells

doi: 10.1101/2023.06.23.546021

Figure Lengend Snippet: A. Brightfield and PI inclusion images of primary keratinocytes upon nigericin (5µg/mL), lasalocid (5µM), gramicidin (1µM) or palytoxin (100pM) 0, 3 and 6 hours post treatment. Arrows indicate lysed cells that are PI negative. Representative data are shown for n = 3. B. Quantification of the percentage of PI positive Cas9 control and NLRP1 KO primary keratinocytes upon lasalocid (5µM) treatment. Cells were imaged at 15 minute intervals for 18 hours. C. Quantification of the percentage of PI positive primary keratinocytes with or without pre-treatment with M443 (1µM) before lasalocid treatment (5µM).Cells were imaged at 15 minute intervals for 18 hours. D. Quantification of the percentage of PI positive primary keratinocytes with or without pre-treatment with emricasan (5µM) before nigericin (5µg/mL) E. Quantification of the percentage of PI positive primary keratinocytes with or without pre-treatment with emricasan (5µM) before palytoxin (100pM) treatment.Cells were imaged at 15 minute intervals for 18 hours. F. Comparison of the inner diameters of the respective channels/pore to the diameters of H2O and ions. Diagram drawn to scale. G. Summary of how nigericin activates NLRP3 and NLRP1 in distinct human cell types.

Article Snippet: The following drugs and chemicals were used as part of this study: Nigericin (Sigma, N7143), Anisomycin (ANS, MCE, #HY18982), Talabostat (VbP, MCE, #HY-13233), MCC950 (MCE, HY-12815A), ATP (Thermofisher Scientific, R0441), Belnacasan (MCE, HY-13205), Emricasan (MCE, HY-10396), Potassium chloride (Sigma Aldrich, P9541), BAPTA (MCE, HY-100168), Harringtonine (HTN, MCE, HY-N0862), Puromycin (PURO, Sigma, #P9620), M443 (MCE, HY-112274), Neflamapimod (p38i) (MCE, HY-10328), Bentamapimod (JNKi) (MCE, HY-14761), Valinomycin (MCE, HY-N6693), Salinomycin (MCE, HY-15597), Monensin (MCE, HY-N0150), Gramicidin (MCE, HY-P0163), Palytoxin (Fujifilm Wako, 165-26141), Nonactin (Sigma Aldrich, N2286), BME-44 (Sigma Aldrich 60397), Lasalocid (MCE, HY-B1071), Emetine (EME, MCE, HY-B1479B).

Techniques:

A. Western blot analysis of primary keratinocytes showing GSDMD (full length and cleaved), GSDME (full length and cleaved), pro-IL1β, cleaved IL1β and GAPDH (loading control) upon overnight treatment with palytoxin (10pM, 100pM or 1000pM), nigericin (5µg/mL) or lasalocid (5µM). Pre-treatment with M443 (1µM) for 15 mins was performed for the relevant conditions. Data are representative of three independent experiments. Immunoblot representative of three replicate experiments. B. Western blot analysis of WT or NLRP1 KO primary keratinocytes showing GSDMD (full length and cleaved), pro-IL1β, cleaved IL1β and HSP70 (loading control) upon overnight treatment with increasing concentration of lasalocid (1µM, 5µM or 10µM), nigericin (5µg/mL) or anisomycin (1uM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments.

Journal: bioRxiv

Article Title: Mechanistic basis for nigericin-induced NLRP1 inflammasome activation in human epithelial cells

doi: 10.1101/2023.06.23.546021

Figure Lengend Snippet: A. Western blot analysis of primary keratinocytes showing GSDMD (full length and cleaved), GSDME (full length and cleaved), pro-IL1β, cleaved IL1β and GAPDH (loading control) upon overnight treatment with palytoxin (10pM, 100pM or 1000pM), nigericin (5µg/mL) or lasalocid (5µM). Pre-treatment with M443 (1µM) for 15 mins was performed for the relevant conditions. Data are representative of three independent experiments. Immunoblot representative of three replicate experiments. B. Western blot analysis of WT or NLRP1 KO primary keratinocytes showing GSDMD (full length and cleaved), pro-IL1β, cleaved IL1β and HSP70 (loading control) upon overnight treatment with increasing concentration of lasalocid (1µM, 5µM or 10µM), nigericin (5µg/mL) or anisomycin (1uM). Data are representative of three independent experiments. Immunoblot representative of three replicate experiments.

Article Snippet: The following drugs and chemicals were used as part of this study: Nigericin (Sigma, N7143), Anisomycin (ANS, MCE, #HY18982), Talabostat (VbP, MCE, #HY-13233), MCC950 (MCE, HY-12815A), ATP (Thermofisher Scientific, R0441), Belnacasan (MCE, HY-13205), Emricasan (MCE, HY-10396), Potassium chloride (Sigma Aldrich, P9541), BAPTA (MCE, HY-100168), Harringtonine (HTN, MCE, HY-N0862), Puromycin (PURO, Sigma, #P9620), M443 (MCE, HY-112274), Neflamapimod (p38i) (MCE, HY-10328), Bentamapimod (JNKi) (MCE, HY-14761), Valinomycin (MCE, HY-N6693), Salinomycin (MCE, HY-15597), Monensin (MCE, HY-N0150), Gramicidin (MCE, HY-P0163), Palytoxin (Fujifilm Wako, 165-26141), Nonactin (Sigma Aldrich, N2286), BME-44 (Sigma Aldrich 60397), Lasalocid (MCE, HY-B1071), Emetine (EME, MCE, HY-B1479B).

Techniques: Western Blot, Concentration Assay

A. Anti-puromycin immunoblots of N/TERT cells subjected to Harringtonine run-off assay. Cells were treated for 3 hours with nonactin (5µM), BME-44 (5µM), nigericin (5µg/mL) or anisomycin (1µM) before harringtonine addition (2µg/mL) for the respective durations. Nascent peptides were then labeled with puromycin (10µg/mL) for 10 mins. Ponceau staining was used as the loading control. Immunoblot representative of three replicate experiments. B. Western blot analysis of PhosTag SDS-page (ZAKɑ) or SDS-page (p-p38, p-JNK and HSP70) of WT or ZAKɑ KO N/TERT cells upon 3 hour treatment with anisomycin (1µM), nigericin (5µg/mL), gramicidin (1µM), lasalocid (5µm) or palytoxin (100pM). Immunoblot representative of three replicate experiments. C. PhosTag SDS-page of NLRP1 KO + NLRP1 DR (a.a.86-254)-GFP N/TERT following 3 hours treatment with ANS (1µM), nigericin (5µg/mL), valinomycin (5µM), salinomycin (5µM), monensin (5µM), gramicidin (1µM), nonactin (5µM), BME-44 (5µM), lasalocid (5µM) or palytoxin (100pM). Immunoblot representative of three replicate experiments. D. Western blot analysis of p-p38, p-JNK, puromycin and HSP70 (loading control) in N/TERTs following pre-treatment with 5µM emricasan and 3 hour treatment with palytoxin 100pM or nigericin (5µg/mL) with or without supplementation of 50mM KCl.

Journal: bioRxiv

Article Title: Mechanistic basis for nigericin-induced NLRP1 inflammasome activation in human epithelial cells

doi: 10.1101/2023.06.23.546021

Figure Lengend Snippet: A. Anti-puromycin immunoblots of N/TERT cells subjected to Harringtonine run-off assay. Cells were treated for 3 hours with nonactin (5µM), BME-44 (5µM), nigericin (5µg/mL) or anisomycin (1µM) before harringtonine addition (2µg/mL) for the respective durations. Nascent peptides were then labeled with puromycin (10µg/mL) for 10 mins. Ponceau staining was used as the loading control. Immunoblot representative of three replicate experiments. B. Western blot analysis of PhosTag SDS-page (ZAKɑ) or SDS-page (p-p38, p-JNK and HSP70) of WT or ZAKɑ KO N/TERT cells upon 3 hour treatment with anisomycin (1µM), nigericin (5µg/mL), gramicidin (1µM), lasalocid (5µm) or palytoxin (100pM). Immunoblot representative of three replicate experiments. C. PhosTag SDS-page of NLRP1 KO + NLRP1 DR (a.a.86-254)-GFP N/TERT following 3 hours treatment with ANS (1µM), nigericin (5µg/mL), valinomycin (5µM), salinomycin (5µM), monensin (5µM), gramicidin (1µM), nonactin (5µM), BME-44 (5µM), lasalocid (5µM) or palytoxin (100pM). Immunoblot representative of three replicate experiments. D. Western blot analysis of p-p38, p-JNK, puromycin and HSP70 (loading control) in N/TERTs following pre-treatment with 5µM emricasan and 3 hour treatment with palytoxin 100pM or nigericin (5µg/mL) with or without supplementation of 50mM KCl.

Article Snippet: The following drugs and chemicals were used as part of this study: Nigericin (Sigma, N7143), Anisomycin (ANS, MCE, #HY18982), Talabostat (VbP, MCE, #HY-13233), MCC950 (MCE, HY-12815A), ATP (Thermofisher Scientific, R0441), Belnacasan (MCE, HY-13205), Emricasan (MCE, HY-10396), Potassium chloride (Sigma Aldrich, P9541), BAPTA (MCE, HY-100168), Harringtonine (HTN, MCE, HY-N0862), Puromycin (PURO, Sigma, #P9620), M443 (MCE, HY-112274), Neflamapimod (p38i) (MCE, HY-10328), Bentamapimod (JNKi) (MCE, HY-14761), Valinomycin (MCE, HY-N6693), Salinomycin (MCE, HY-15597), Monensin (MCE, HY-N0150), Gramicidin (MCE, HY-P0163), Palytoxin (Fujifilm Wako, 165-26141), Nonactin (Sigma Aldrich, N2286), BME-44 (Sigma Aldrich 60397), Lasalocid (MCE, HY-B1071), Emetine (EME, MCE, HY-B1479B).

Techniques: Western Blot, Labeling, Staining, SDS Page