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MB Sample ID: SA237439
| Local Sample ID: | LPS_ATP_2 |
| Subject ID: | SU002468 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002468 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| LPS_ATP_2 | SA237439 | FL029416 | LPS+ATP | Treatment |
Collection:
| Collection ID: | CO002461 |
| Collection Summary: | Mouse bone marrow was cultured in low glucose DMEM supplemented with 30% L929 cell-conditioned medium, 20% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin for 6-7 days until the cells reached confluence. BMDMs were then reseeded in culture dishes overnight in RPMI-1640 medium containing 1% Nutridoma-SP medium (Sigma-Aldrich). LPS-primed BMDMs were stimulated with 5 mM ATP in the presence or absence of 10 uM JX06 for 30 min. Unstimulated macrophages were used as control.13C tracing started by replacing medium with glucose-free medium supplemented with 2.06 g/L [U-13C]-glucose (Cayman Chemical) for 90 min. Cells were washed with ice-cold saline and intracellular metabolites were extracted using cold methanol and H2O (80:20; HPLC Grade; Sigma-Aldrich). 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. |
| Sample Type: | Macrophages |
| Collection Method: | Cells were washed with ice-cold saline and intracellular metabolites were extracted using cold methanol and H2O (80:20; HPLC Grade; Sigma-Aldrich). The samples were pre-dissolved into 40 µL solution (LC-water, methanol, Acetonitrile 2:1:1) before injection. |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002480 |
| Treatment Summary: | LPS-primed BMDMs were stimulated with or without 5 mM ATP in the presence or absence of 10 uM JX06 for 30 min. 13C tracing started by replacing medium with glucose-free medium supplemented with 2.06 g/L [U-13C]-glucose (Cayman Chemical) for 90 min. |
| Treatment: | in vitro cell culture treatment |
| Treatment Compound: | LPS, ATP, JX06 |
Sample Preparation:
| Sampleprep ID: | SP002474 |
| Sampleprep Summary: | Macrophages were lysed, and polar metabolites were extracted using methanol and H2O (80:20; HPLC Grade; Sigma-Aldrich) (Liu et al., 2015). Extracts were dried in a vacuum concentrator at room temperature and stored at -80 freezer. |
Combined analysis:
| Analysis ID | AN003877 | AN003878 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 | Thermo Dionex Ultimate 3000 |
| Column | Waters XBridge Amide (100 x 4.6mm,3.5um) | Waters XBridge Amide (100 x 4.6mm,3.5um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Plus Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | Fractional enrichment | Fractional enrichment |
Chromatography:
| Chromatography ID: | CH002873 |
| Chromatography Summary: | Ultimate 3000 UHPLC (Dionex) is coupled to Q Exactive Plus-Mass spectrometer (QE-MS, Thermo Scientific) for metabolite profiling. |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Waters XBridge Amide (100 x 4.6mm,3.5um) |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003618 |
| Analysis ID: | AN003877 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | For each sample, 3 µL solution was injected into the LC-MS for analysis. The HPLC analysis of the isotope-labeled samples was performed using Ultimate 3000 UHPLC (Dionex) as described (Duan et al., 2022). The mass spectrometry analysis was performed using Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). The mass spectrometers were equipped with a HESI probe and operated in the positive/negative switching mode. When Q Exactive Plus mass spectrometer was used, the relevant parameters were as listed: heater temperature, 120 °C; sheath gas, 30; auxiliary gas, 10; sweep gas, 3; spray voltage, 3.0 kV; capillary temperature, 320°C; S-lens, 55. The resolution was set at 70,000 (at m/z 200). Maximum injection time (max IT) was set at 200 ms and automated gain control (AGC) was set at 3 × 106. The LC-MS peak extraction and integration of the raw data were performed using commercially available software Sieve 2.0 (Thermo Fisher Scientific). The integrated peak area was used to calculate 13C enrichment. Natural abundance correction was performed using software R with Bioconductor R package IsoCorrectoR. |
| Ion Mode: | POSITIVE |
| MS ID: | MS003619 |
| Analysis ID: | AN003878 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | For each sample, 3 µL solution was injected into the LC-MS for analysis. The HPLC analysis of the isotope-labeled samples was performed using Ultimate 3000 UHPLC (Dionex) as described (Duan et al., 2022). The mass spectrometry analysis was performed using Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). The mass spectrometers were equipped with a HESI probe and operated in the positive/negative switching mode. When Q Exactive Plus mass spectrometer was used, the relevant parameters were as listed: heater temperature, 120 °C; sheath gas, 30; auxiliary gas, 10; sweep gas, 3; spray voltage, 3.0 kV; capillary temperature, 320°C; S-lens, 55. The resolution was set at 70,000 (at m/z 200). Maximum injection time (max IT) was set at 200 ms and automated gain control (AGC) was set at 3 × 106. The LC-MS peak extraction and integration of the raw data were performed using commercially available software Sieve 2.0 (Thermo Fisher Scientific). The integrated peak area was used to calculate 13C enrichment. Natural abundance correction was performed using software R with Bioconductor R package IsoCorrectoR. |
| Ion Mode: | NEGATIVE |
