TNF was from R&D Systems. LPS (L2630), Antimycin A, CCCP, Rotenone, Mito-TEMPO, bafilomycin A1, MG132, and CHX were from Sigma-Aldrich. ATP was from ENZO Life Sciences. TMRE, MitoTracker Deep Red, MitoTracker Green and MitoSox Red were from Life technologies. HBSS was from Cellgro. The ProcartaPlex Mouse Basic kit, mouse IL-1β Simplex and IL-18 Simplex were from eBioscience. Monoclonal antibodies used in this study are listed in Supplementary Table 1 . The mCherry-Parkin plasmid was a gift from Richard Youle (Addgene plasmid # 23956)1 (link),2 (link). The following siRNA oligos were from Thermo Scientific (Dharmacon products): control siRNA (D-001210-02); ARF siRNA # 1 (5′-AGGUGAUGAUGAUGGGCAATT-3′); ARF siRNA # 2 (5′-GGUCGCAGGUUCUUGGUCATT-3′); p62 siRNA (L-047628-01); Jnk2 siRNA (5′-CCGCAGAGUUCAUGAAGAATT-3′); Jnk1 siRNA (5′-UGAUUCAGAUGGAGUUAGATT-3′); Beclin 1 (5′-CAGUUUGGCACAAUCAAUATT-3′); ATG5 siRNA (5′-CAUCAACCGGAAACUCAUTT-3′).
Adenosine triphosphate (atp)
Manufactured by Enzo Life Sciences
Sourced in China, United States
ATP is a nucleoside triphosphate that serves as a coenzyme in numerous biological processes. It is the primary energy currency of the cell, participating in a variety of metabolic pathways.
Automatically generated - may contain errors
Lab products found in correlation
Lps l2630, by Merck Group (3 mentions)
Tumor necrosis factor (tnf), by R&D Systems (2 mentions)
Procartaplex mouse basic kit, by Thermo Fisher Scientific (2 mentions)
Control sirna, by Thermo Fisher Scientific (2 mentions)
Mitosox red, by Thermo Fisher Scientific (2 mentions)
Mitotracker green, by Thermo Fisher Scientific (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Mouse il 1β simplex, by Thermo Fisher Scientific (2 mentions)
Il 18 simplex, by Thermo Fisher Scientific (2 mentions)
Mitotempo, by Merck Group (2 mentions)
Hanks balanced salt solution (hbss), by Corning (2 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (2 mentions)
Mg132, by Merck Group (2 mentions)
Bafilomycin a1, by Merck Group (2 mentions)
Antimycin a, by Merck Group (2 mentions)
Rotenone, by Merck Group (2 mentions)
Nigericin, by Merck Group (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Glyburide, by Selleck Chemicals (1 mentions)
Lipopolysaccharides (lps), by Selleck Chemicals (1 mentions)
8 protocols using adenosine triphosphate (atp)
1
Mitochondrial Dynamics in Immune Regulation
2
Neospora caninum Infection and NLRP3 Inflammasome
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At 3 h prior to infection, the medium was changed to complete medium (RPMI) plus 1% fetal bovine serum with or without 100 ng/ml ultrapure lipopolysaccharide (LPS; Sigma, Shanghai, China). The PMs were stimulated with N. caninum for the indicated times (1–24 h) at various multiplicities of infection (MOI = 1:1, 3:1, and 5:1). PMs treated with medium alone or LPS were used as negative controls, and cells treated with ATP (5 mM, 30 min; Sigma, Shanghai, China) were used as positive controls, as extracellular ATP is a conventional NLRP3 inflammasome agonist [14 (link)].
To monitor the role of the inflammasome in response to N. caninum infection in PMs, PMs were pre-treated with 100 μM Ac-YVAD-CHO (an inhibitor of caspase-1 and -4; Enzo Life Science, Lausen, Switzerland), 100 μM zVAD-fmk (an inhibitor of pan-caspase; Selleck, Shanghai, China) or 100 μM glyburide (an inhibitor of NLRP3 by inhibiting K+ efflux; Selleck, Shanghai, China) for 45 min before stimulation, and the PMs were then stimulated with LPS priming plus N. caninum for 12 h at an MOI of 3:1 (parasite:cell), and PMs cultured with 0.2% DMSO were used as a negative control.
To monitor the role of the inflammasome in response to N. caninum infection in PMs, PMs were pre-treated with 100 μM Ac-YVAD-CHO (an inhibitor of caspase-1 and -4; Enzo Life Science, Lausen, Switzerland), 100 μM zVAD-fmk (an inhibitor of pan-caspase; Selleck, Shanghai, China) or 100 μM glyburide (an inhibitor of NLRP3 by inhibiting K+ efflux; Selleck, Shanghai, China) for 45 min before stimulation, and the PMs were then stimulated with LPS priming plus N. caninum for 12 h at an MOI of 3:1 (parasite:cell), and PMs cultured with 0.2% DMSO were used as a negative control.
3
THP1 Cell Inflammasome Activation
4
In Vitro SUMOylation of PVR
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A recombinant full-lenght human form of PVR (Abnova, H00005817) was used as substrate for in vitro SUMOylation. The reaction was carried out using 200 ng of the target protein in a 20 μL reaction volume containing E1 and E2 enzymes involved in substrate SUMOylation, SUMO 1/2/3 proteins and SUMOylation buffer for 1 hour at 37 °C in the presence of ATP, according to manufacturer’s instructions (Enzo Life Sciences). Negative control reaction omitting Mg-ATP cofactors (required for E1 activation) was performed. After in vitro SUMOylation samples were eluted with SDS-sample buffer and resolved by SDS/PAGE.
5
Mitochondrial Dynamics in Immune Regulation
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TNF was from R&D Systems. LPS (L2630), Antimycin A, CCCP, Rotenone, Mito-TEMPO, bafilomycin A1, MG132, and CHX were from Sigma-Aldrich. ATP was from ENZO Life Sciences. TMRE, MitoTracker Deep Red, MitoTracker Green and MitoSox Red were from Life technologies. HBSS was from Cellgro. The ProcartaPlex Mouse Basic kit, mouse IL-1β Simplex and IL-18 Simplex were from eBioscience. Monoclonal antibodies used in this study are listed in Supplementary Table 1 . The mCherry-Parkin plasmid was a gift from Richard Youle (Addgene plasmid # 23956)1 (link),2 (link). The following siRNA oligos were from Thermo Scientific (Dharmacon products): control siRNA (D-001210-02); ARF siRNA # 1 (5′-AGGUGAUGAUGAUGGGCAATT-3′); ARF siRNA # 2 (5′-GGUCGCAGGUUCUUGGUCATT-3′); p62 siRNA (L-047628-01); Jnk2 siRNA (5′-CCGCAGAGUUCAUGAAGAATT-3′); Jnk1 siRNA (5′-UGAUUCAGAUGGAGUUAGATT-3′); Beclin 1 (5′-CAGUUUGGCACAAUCAAUATT-3′); ATG5 siRNA (5′-CAUCAACCGGAAACUCAUTT-3′).
6
In vitro ubiquitination assay of IRGM1
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His-IRGM1 was expressed in E.coli and purified with Ni2+-NTA (GE Healthcare). His-IRGM1 was incubated with 200 ng E1 (UBE1), 500 ng E2 (UbcH5c), 10 μg His-Ub and 2 mM ATP (Enzo life sciences). The reaction was performed in the absence and presence of Flag-CUL4B co-IP products and incubated at 37 °C for 1 h. Samples were quenched in 6 M guanidinium-HCl (pH=8) containing 5 mM NEM. His-ubiquitinated proteins were pulled down with Ni2+-NTA (GE Healthcare), washed and eluted in sample buffers as described previously [27 (link)]. The mixture was then boiled in loading dye at 95 °C for 10 mins to disrupt the protein-protein interactions, followed by Western blot.
7
miR-221-5p Regulation of JNK2 Signaling
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LPS (L2630), β-actin antibody (AC-15), and actinomycin D were from Sigma-Aldrich. ATP was from ENZO Life Sciences. JNK2 antibody was from Cell Signaling Technology (Cat # 4672). AGO2 antibody was from abcam (ab186733). Mouse miRIDIAN miR-221-5p mimic, miRIDIAN miRNA mimic negative control, miRIDIAN mouse miR-221-5p hairpin inhibitor, and miRIDIAN miNA hairpin inhibitor negative control were from Horizon Discovery. Ad/JNK2 was generated by ViraQuest, Inc. by cloning pCDNA3 Flag Jnk2a2 (Addgene, Item ID 13755) into the pVQAd CMV K-NpA vector. Ad/null was from ViraQuest, Inc. miR-221-5p inhibitor and inhibitor control, and miR-221-5p mimic and mimic control were from Horizon Discovery.
8
Inflammasome Activation and IL-1β Secretion
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PMA (Sigma-Aldrich, St. Louis, MO, USA), nigericin (Sigma-Aldrich), glyburide (Sigma-Aldrich), ATP (Enzo lifesciences, Farmingdale, NY, USA), KCl (Sigma-Aldrich), TAPI-2 (EMD chemicals, Gibbstown, NJ, USA), AG-825 (EMD chemicals), recombinant human IL-1β (rhIL-1β, R&D systems, Minneapolis, MN, USA) and IL-1βRA (rhIL-1β, R&D systems) were purchased from its respective vendors. The following secondary antibodies were used to detect protein expression: anti-rabbit HER2 (Santacruz biotechnology, Santa Cruz, CA, USA), anti-rabbit pHER2 (Santacruz biotechnology), anti-mouse NRG-1(Neo markers, Fremont, CA, USA) primary, anti-mouse (Cell Signaling Technology, Danvers, MA, USA) and anti-rabbit (Cell Signaling Technology).
Treatment with inflammasome stimulators or inhibitors THP-1 cells were differentiated by adding 0.5 µg/ml of PMA to the medium for 3 hours, then the medium was replaced with RPMI plus 0.1% serum overnight. After 24 hours, cells were briefly washed with PBS. Then, 0.5 µg/ml of nigericin or 0.25 mM ATP, in the presence or absence of 1.25 µg/ml glyburide or 130 mM KCl, was added to the medium for 1 hour. The activation of the inflammasome was confirmed by measuring the IL-1β secretion, and the supernatant was added to 80% confluent epithelial or endothelial cells for up to 5% of the mentioned time.
Treatment with inflammasome stimulators or inhibitors THP-1 cells were differentiated by adding 0.5 µg/ml of PMA to the medium for 3 hours, then the medium was replaced with RPMI plus 0.1% serum overnight. After 24 hours, cells were briefly washed with PBS. Then, 0.5 µg/ml of nigericin or 0.25 mM ATP, in the presence or absence of 1.25 µg/ml glyburide or 130 mM KCl, was added to the medium for 1 hour. The activation of the inflammasome was confirmed by measuring the IL-1β secretion, and the supernatant was added to 80% confluent epithelial or endothelial cells for up to 5% of the mentioned time.
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