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MB Sample ID: SA237423
| Local Sample ID: | WT-JX-3 |
| Subject ID: | SU002467 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Gender: | Male and female |
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Subject:
| Subject ID: | SU002467 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Gender: | Male and female |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| WT-JX-3 | SA237423 | FL029412 | WT | Genotype |
| WT-JX-3 | SA237423 | FL029412 | LPS+JX06+ATP | Treatment |
Collection:
| Collection ID: | CO002460 |
| Collection Summary: | Macrophages were lysed, and polar metabolites were extracted using methanol and H2O (80:20; HPLC Grade; Sigma-Aldrich). Briefly, After treatment, immediately aspirate medium at room temperature. Immediately place the plate on dry ice, and add 1 mL 80% methanol/water (both HPLC grade) (pre-cooled in -80oC for at least 1hr).Remove the plate from -80oC freezer and put it on dry ice, scrape cells into extraction solvent. Transfer the whole cell extract to a new Eppendorf tube placed on ice. Centrifuge at 20 000 rcf for 10 min, 4oC.Transfer the supernatant into two tubes and dry with a speed vacuum at room temperature. |
| Sample Type: | Macrophages |
| Storage Conditions: | Described in summary |
Treatment:
| Treatment ID: | TR002479 |
| Treatment Summary: | WT and GSDMD KO bone marrow derived macrophages were first primed with 300 ng/ml LPS (E. coli 0111; B4, Sigma-Aldrich) before stimulated with or without 5 mM ATP (Sigma-Aldrich) for 30 min in the presence or absence of 10 uM JX06. Here, we show that pharmacological or genetic inhibition of pyruvate dehydrogenase kinase (PDHK) significantly attenuates NLRP3 inflammasome activation in murine and human macrophages and septic mice by lowering caspase-1 cleavage and IL-1beta secretion. Inhibiting PDHK reverses NLRP3 inflammasome-induced metabolic reprogramming, enhances autophagy, promotes mitochondrial fusion over fission, preserves cristae ultrastructure, and attenuates mitochondrial ROS production. The suppressive effect of PDHK inhibition on the NLRP3 inflammasome is independent of its canonical role as a pyruvate dehydrogenase regulator. We suggest that PDHK inhibition improves mitochondrial fitness by reversing NLRP3 inflammasome activation in acutely inflamed macrophages. |
| Treatment: | In vitro culture |
| Treatment Compound: | LPS, ATP, JX06 |
Sample Preparation:
| Sampleprep ID: | SP002473 |
| Sampleprep Summary: | Macrophages were lysed, and polar metabolites were extracted using methanol and H2O (80:20; HPLC Grade; Sigma-Aldrich). Briefly, After treatment, immediately aspirate medium at room temperature. Immediately place the plate on dry ice, and add 1 mL 80% methanol/water (both HPLC grade) (pre-cooled in -80oC for at least 1hr).Remove the plate from -80oC freezer and put it on dry ice, scrape cells into extraction solvent. Transfer the whole cell extract to a new Eppendorf tube placed on ice. Centrifuge at 20 000 rcf for 10 min, 4oC.Transfer the supernatant into two tubes and dry with a speed vacuum at room temperature. The dried metabolites were stored at -80 freezer before analysis. |
| Processing Storage Conditions: | Described in summary |
| Extract Storage: | Described in summary |
Combined analysis:
| Analysis ID | AN003875 | AN003876 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Ion pair | Unspecified |
| Chromatography system | Shimadzu Nexera X2 | Shimadzu Nexera X2 |
| Column | Agilent Zorbax Eclipse Plus C18 (100 x 2.1mm, 1.8 um) | Restek Ultra PFPP (150 x 2.1mm,3um) |
| MS Type | ESI | ESI |
| MS instrument type | Triple quadrupole | Triple quadrupole |
| MS instrument name | Shimadzu Nexera X2 | Shimadzu Nexera X2 |
| Ion Mode | NEGATIVE | NEGATIVE |
| Units | Peak area ratio | Peak area ratio |
Chromatography:
| Chromatography ID: | CH002871 |
| Chromatography Summary: | 500 μL of cell extract and 20 μL of MES (Thermo Fisher Scientific, Waltham, MA, USA) internal standard solution (10 ng/μL) were combined and mixed thoroughly through vortexing. The mixture was then dried under vacuum and reconstituted for analysis in 100 μL of ultrapure water (Optima, Thermo Fisher Scientific, Waltham, MA, USA). The analysis was performed on a Shimadzu Nexera UHPLC system coupled with a Shimadzu LCMS-8050 triple-quadrupole mass spectrometer (Kyoto, Japan). Two LC-MS/MS methods were employed to measure the targets. Ion-Pairing Separation was performed at 0.3 ml/min on a Zorbax Eclipse Plus C18 column (1.8 μm, 2.1 x 100 mm; Agilent, Santa Clara, CA USA). Mobile phase A consisted of ultrapure water (Optima, Thermo Fisher Scientific, Waltham, MA, USA) with 10 mM ammonium acetate (J.T. Baker, Thermo Fisher Scientific, Waltham, MA, USA) and 10 mM tributylamine (Acros Organics, Thermo Fisher Scientific, Fair Lawn NJ, USA) and mobile phase B consisted of methanol (Optima, Thermo Fisher Scientific, Waltham, MA, USA). The separation used the following gradient profile: 2 minutes at 0% B, a ramp to 25% B at 8 minutes, another ramp to 98%B at 12 minutes, a 3 minute hold until 15 minutes, and then a drop back to 0% B at 15.1 minutes and allowed to equilibrate there until 25 minutes. All analytes were monitored in negative mode. |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Agilent Zorbax Eclipse Plus C18 (100 x 2.1mm, 1.8 um) |
| Flow Gradient: | The separation used the following gradient profile: 2 minutes at 0% B, a ramp to 25% B at 8 minutes, another ramp to 98%B at 12 minutes, a 3 minute hold until 15 minutes, and then a drop back to 0% B at 15.1 minutes and allowed to equilibrate there until 25 minutes. |
| Solvent A: | 100% water; 10 mM ammonium acetate; 10 mM tributylamine |
| Solvent B: | 100% methanol |
| Chromatography Type: | Ion pair |
| Chromatography ID: | CH002872 |
| Chromatography Summary: | PFPP A gradient separation on a Restek Ultra PFPP column (150 x 2.1 mm, 3 μm; Restek, Bellefonte, PA) was used to separate the analytes. Mobile phase A consisted of 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 100% acetonitrile. The gradient began with 2 minutes at 0% B followed by a ramp to 25% B between 2 and 5 minutes, another ramp to 35% B between 5 and 11 minutes, a final increase from 35% to 95% B between 11 and 15 minutes, a hold at 95% B from 15 to 20 minutes, then a decrease to 0% B between 20 and 20.10 minutes, and a final hold at 0% B from 20.10 to 25 minutes. The flow rate to achieve separation was 0.25 ml/min. Ionization in the negative ESI mode occurred in the DUIS source with the following conditions: nebulizing gas flow of 2 L/min, heating gas flow of 5 L/min, interface temperature of 350°C, DL temperature of 250°C, heat block temperature of 400°C, and a drying gas flow of 15 L/min. |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Restek Ultra PFPP (150 x 2.1mm,3um) |
| Flow Gradient: | The gradient began with 2 minutes at 0% B followed by a ramp to 25% B between 2 and 5 minutes, another ramp to 35% B between 5 and 11 minutes, a final increase from 35% to 95% B between 11 and 15 minutes, a hold at 95% B from 15 to 20 minutes, then a decrease to 0% B between 20 and 20.10 minutes, and a final hold at 0% B from 20.10 to 25 minutes. |
| Flow Rate: | 0.25 ml/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | Unspecified |
MS:
| MS ID: | MS003616 |
| Analysis ID: | AN003875 |
| Instrument Name: | Shimadzu Nexera X2 |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | The analysis was performed on a Shimadzu Nexera UHPLC system coupled with a Shimadzu LCMS-8050 triple-quadrupole mass spectrometer (Kyoto, Japan). Two LC-MS/MS methods were employed to measure the targets. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS003617 |
| Analysis ID: | AN003876 |
| Instrument Name: | Shimadzu Nexera X2 |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | The analysis was performed on a Shimadzu Nexera UHPLC system coupled with a Shimadzu LCMS-8050 triple-quadrupole mass spectrometer (Kyoto, Japan). Two LC-MS/MS methods were employed to measure the targets. |
| Ion Mode: | NEGATIVE |
