#METABOLOMICS WORKBENCH yaqiu_20230616_110728 DATATRACK_ID:4095 STUDY_ID:ST002738 ANALYSIS_ID:AN004441 PROJECT_ID:PR001703 VERSION 1 CREATED_ON 11-18-2025 #PROJECT PR:PROJECT_TITLE Innate immune and metabolic signaling retain damaged mitochondria at cell PR:PROJECT_TITLE membranes for mitoxyperilysis PR:PROJECT_SUMMARY Innate immune activation coupled with metabolic disruptions play critical roles PR:PROJECT_SUMMARY in many diseases, often leading to mitochondrial dysfunction and oxidative PR:PROJECT_SUMMARY stress that drive pathogenesis. However, mechanistic regulation under these PR:PROJECT_SUMMARY conditions remains poorly defined. Here, we report a distinct lytic cell death PR:PROJECT_SUMMARY mechanism induced by innate immune signaling and metabolic disruption, PR:PROJECT_SUMMARY independent of caspase activity and previously described pyroptosis, PANoptosis, PR:PROJECT_SUMMARY necroptosis, ferroptosis, and oxeiptosis. Instead, mitochondria undergoing PR:PROJECT_SUMMARY BAX/BAK1/BID-dependent oxidative stress maintained prolonged plasma membrane PR:PROJECT_SUMMARY contact, leading to local oxidative damage, a process we termed mitoxyperiosis. PR:PROJECT_SUMMARY This process then caused membrane lysis and cell death, mitoxyperilysis. mTORC2 PR:PROJECT_SUMMARY regulated the cell death, and mTOR inhibition restored cytoskeletal activity for PR:PROJECT_SUMMARY lamellipodia retractions to mobilize mitochondria away from the membrane, PR:PROJECT_SUMMARY preserving integrity. Activating this pathway in vivo regressed tumors in an PR:PROJECT_SUMMARY mTORC2-dependent manner. Overall, our results identify a lytic cell death PR:PROJECT_SUMMARY modality in response to the synergism of innate immune signaling and metabolic PR:PROJECT_SUMMARY disruption. PR:INSTITUTE St Jude Children's Research Hospital PR:LAST_NAME Wang PR:FIRST_NAME Yaqiu PR:ADDRESS 262 Danny Thomas Pl, Memphis, Tennessee, 38105, USA PR:EMAIL yaqiu.wang@stjude.org PR:PHONE +1-901-595-3477 PR:DOI http://dx.doi.org/10.21228/M8K99P #STUDY ST:STUDY_TITLE Innate immune and metabolic signaling retain damaged mitochondria at cell ST:STUDY_TITLE membranes for mitoxyperilysis ST:STUDY_SUMMARY Innate immune activation coupled with metabolic disruptions play critical roles ST:STUDY_SUMMARY in many diseases, often leading to mitochondrial dysfunction and oxidative ST:STUDY_SUMMARY stress that drive pathogenesis. However, mechanistic regulation under these ST:STUDY_SUMMARY conditions remains poorly defined. Here, we report a distinct lytic cell death ST:STUDY_SUMMARY mechanism induced by innate immune signaling and metabolic disruption, ST:STUDY_SUMMARY independent of caspase activity and previously described pyroptosis, PANoptosis, ST:STUDY_SUMMARY necroptosis, ferroptosis, and oxeiptosis. Instead, mitochondria undergoing ST:STUDY_SUMMARY BAX/BAK1/BID-dependent oxidative stress maintained prolonged plasma membrane ST:STUDY_SUMMARY contact, leading to local oxidative damage, a process we termed mitoxyperiosis. ST:STUDY_SUMMARY This process then caused membrane lysis and cell death, mitoxyperilysis. mTORC2 ST:STUDY_SUMMARY regulated the cell death, and mTOR inhibition restored cytoskeletal activity for ST:STUDY_SUMMARY lamellipodia retractions to mobilize mitochondria away from the membrane, ST:STUDY_SUMMARY preserving integrity. Activating this pathway in vivo regressed tumors in an ST:STUDY_SUMMARY mTORC2-dependent manner. Overall, our results identify a lytic cell death ST:STUDY_SUMMARY modality in response to the synergism of innate immune signaling and metabolic ST:STUDY_SUMMARY disruption. ST:INSTITUTE St Jude Children's Research Hospital ST:LAST_NAME Wang ST:FIRST_NAME Yaqiu ST:ADDRESS 262 Danny Thomas Pl ST:EMAIL yaqiu.wang@stjude.org ST:PHONE 901-595--3477 ST:SUBMIT_DATE 2023-06-16 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:SPECIES_GROUP Mammals #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_00_Neg Treatment:BLANK RAW_FILE_NAME=YW_01272023_Metabo_00_Neg.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_00_Pos Treatment:BLANK RAW_FILE_NAME=YW_01272023_Metabo_00_Pos.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_10_Neg Treatment:CS+LPS_WT RAW_FILE_NAME=YW_01272023_Metabo_10_Neg.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_10_Pos Treatment:CS+LPS_WT RAW_FILE_NAME=YW_01272023_Metabo_10_Pos.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_11_Neg Treatment:CS+LPS_WT RAW_FILE_NAME=YW_01272023_Metabo_11_Neg.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_11_Pos Treatment:CS+LPS_WT RAW_FILE_NAME=YW_01272023_Metabo_11_Pos.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_12_Neg Treatment:CS+LPS_WT RAW_FILE_NAME=YW_01272023_Metabo_12_Neg.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_12_Pos Treatment:CS+LPS_WT RAW_FILE_NAME=YW_01272023_Metabo_12_Pos.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_07_Neg Treatment:CS_WT RAW_FILE_NAME=YW_01272023_Metabo_07_Neg.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_07_Pos Treatment:CS_WT RAW_FILE_NAME=YW_01272023_Metabo_07_Pos.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_08_Neg Treatment:CS_WT RAW_FILE_NAME=YW_01272023_Metabo_08_Neg.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_08_Pos Treatment:CS_WT RAW_FILE_NAME=YW_01272023_Metabo_08_Pos.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_09_Neg Treatment:CS_WT RAW_FILE_NAME=YW_01272023_Metabo_09_Neg.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_09_Pos Treatment:CS_WT RAW_FILE_NAME=YW_01272023_Metabo_09_Pos.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_04_Neg Treatment:LPS_WT RAW_FILE_NAME=YW_01272023_Metabo_04_Neg.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_04_Pos Treatment:LPS_WT RAW_FILE_NAME=YW_01272023_Metabo_04_Pos.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_05_Neg Treatment:LPS_WT RAW_FILE_NAME=YW_01272023_Metabo_05_Neg.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_05_Pos Treatment:LPS_WT RAW_FILE_NAME=YW_01272023_Metabo_05_Pos.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_06_Neg Treatment:LPS_WT RAW_FILE_NAME=YW_01272023_Metabo_06_Neg.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_06_Pos Treatment:LPS_WT RAW_FILE_NAME=YW_01272023_Metabo_06_Pos.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_01_Neg Treatment:Media_WT RAW_FILE_NAME=YW_01272023_Metabo_01_Neg.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_01_Pos Treatment:Media_WT RAW_FILE_NAME=YW_01272023_Metabo_01_Pos.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_02_Neg Treatment:Media_WT RAW_FILE_NAME=YW_01272023_Metabo_02_Neg.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_02_Pos Treatment:Media_WT RAW_FILE_NAME=YW_01272023_Metabo_02_Pos.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_03_Neg Treatment:Media_WT RAW_FILE_NAME=YW_01272023_Metabo_03_Neg.mzXML SUBJECT_SAMPLE_FACTORS - YW_01272023_Metabo_03_Pos Treatment:Media_WT RAW_FILE_NAME=YW_01272023_Metabo_03_Pos.mzXML #COLLECTION CO:COLLECTION_SUMMARY Ten million BMDMs were stimulated as indicated in 100 mm plates for 9 h. The CO:COLLECTION_SUMMARY medium was removed by aspiration, and the cells were washed once with ice-cold CO:COLLECTION_SUMMARY saline. Next, the cells were carefully harvested using a plastic scraper in 1.5 CO:COLLECTION_SUMMARY ml ice-cold saline. The cells were centrifuged at 150 × g for 2 min at room CO:COLLECTION_SUMMARY temperature, and the cell pellets were flash-frozen in liquid nitrogen and then CO:COLLECTION_SUMMARY stored at −80°C for a later extraction of metabolites. CO:SAMPLE_TYPE Cultured cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Treatment of cells were done as indicated in the study design table. Four types TR:TREATMENT_SUMMARY of treatment were used in WT cells. "Media_WT", "LPS_WT", "CS_WT", "CS+LPS_WT" TR:TREATMENT_SUMMARY denote the WT cells receiving Media, LPS(lipopolysaccharide), CS(starvation) and TR:TREATMENT_SUMMARY CS+LPS treatment, respectively. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Extraction of hydrophilic metabolites To extract the molecules with different SP:SAMPLEPREP_SUMMARY polarity, an adapted three-phase solvent system was utilized to obtain total SP:SAMPLEPREP_SUMMARY hydrophilic metabolites and lipids78. Briefly, the cell pellets were resuspended SP:SAMPLEPREP_SUMMARY with 150 µL of saline, then 1.2 mL of chloroform/methanol/water (3:4:1, v/v/v) SP:SAMPLEPREP_SUMMARY was added and homogenized using a Bead Ruptor Elite (OMNI international) for 30 SP:SAMPLEPREP_SUMMARY s at 8 m/s. The homogenate was allowed to rest at 4ºC for 30 s and then SP:SAMPLEPREP_SUMMARY centrifuged for 10 min at 21,000 g at 4ºC. After centrifugation, the upper SP:SAMPLEPREP_SUMMARY aqueous phase was transferred into a new tube, frozen on dry ice, and then SP:SAMPLEPREP_SUMMARY lyophilized. The dried extracts containing hydrophilic metabolites were SP:SAMPLEPREP_SUMMARY dissolved using 40 µL of water: acetonitrile (95:5, v/v) supplemented with 10 SP:SAMPLEPREP_SUMMARY mM ammonium acetate, then transferred into autosampler vials, and 4 µL per SP:SAMPLEPREP_SUMMARY injection was analyzed by LC-MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY A Vanquish Horizon UHPLC (Thermo Fisher Scientific) was used for the LC CH:CHROMATOGRAPHY_SUMMARY separations, using stepped gradient conditions as follows: 0–16.5 min, 1 to CH:CHROMATOGRAPHY_SUMMARY 50% B; 16.5–18 min, 50 to 99% B; 18–36 min, 99% B; 36–39 min, 99 to 1% B; CH:CHROMATOGRAPHY_SUMMARY 39–45 min, 1% B. Mobile phase A was water supplemented with 10 mM ammonium CH:CHROMATOGRAPHY_SUMMARY acetate. Mobile phase B was acetonitrile. The column used was an xBride BEH CH:CHROMATOGRAPHY_SUMMARY Amide Column (2.1 mm x 150 mm, 2.5 μm) (Waters Corp.), operated at 40°C. The CH:CHROMATOGRAPHY_SUMMARY flow rate was 100 μL/min, and the injection volume was 4 μL. The collected CH:CHROMATOGRAPHY_SUMMARY positive and negative mode data were analyzed separately. CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters XBridge BEH Amide (150 x 2.1mm, 2.5um) CH:COLUMN_TEMPERATURE 40 CH:FLOW_GRADIENT 0–16.5 min, 1 to 50% B; 16.5–18 min, 50 to 99% B; 18–36 min, 99% B; CH:FLOW_GRADIENT 36–39 min, 99 to 1% B; 39–45 min, 1% B. CH:FLOW_RATE 100 μL/min CH:SOLVENT_A water supplemented with 10 mM ammonium acetate CH:SOLVENT_B acetonitrile CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS A Thermo Fisher Scientific Q Exactive hybrid quadrupole-Orbitrap mass MS:MS_COMMENTS spectrometer (QE-MS) (Thermo Fisher Scientific) equipped with a HESI-II probe MS:MS_COMMENTS was employed as the detector. For each sample, two chromatographic runs were MS:MS_COMMENTS carried out subsequently, acquiring separately data for negative and positive MS:MS_COMMENTS ions. The QE-MS was operated using a data-dependent LC-MS/MS method (Top10 MS:MS_COMMENTS dd-MS2) for both, positive and negative ion modes. The mass spec was operated at MS:MS_COMMENTS a resolution of 140,000 (FWHM, at m/z 200), AGC targeted of 3 × 106, Max MS:MS_COMMENTS injection time 100 msec. The instrument’s operating conditions were: scan MS:MS_COMMENTS range 60–900 m/z, sheath gas flow 20, aux gas flow 5, sweep gas 1, spray MS:MS_COMMENTS voltage 3.6 kV for positive mode and 2.5 kV for negative mode, capillary MS:MS_COMMENTS temperature 320ºC, S-lenses RF level 55, aux gas heater 320ºC. For the Top10 MS:MS_COMMENTS dd-MS2 conditions a resolution of 35,000 was used, AGC targeted of 1 × 105, max MS:MS_COMMENTS injection time 100 msec, MS2 isolation width 1.0 m/z, NCE 35. The collected data MS:MS_COMMENTS from negative and positive mode were analyzed separately in Compound Discoverer MS:MS_COMMENTS 3.3 (CD3.3) (Thermo Fisher Scientific). A pre-defined workflow from the software MS:MS_COMMENTS was employed: Untargeted Metabolomics with statistics, Detect Unknowns with ID MS:MS_COMMENTS using online databases and mzLogic. The spectra alignment node used the first MS:MS_COMMENTS sample as the reference file. The compound detection option was set for a MS:MS_COMMENTS signal-to-noise ratio ≥ 1.5, min peak intensity 1 × 106, parent ion mass MS:MS_COMMENTS tolerance of ±5 ppm, RT tolerance of 0.25 min, and preferred fragments data MS:MS_COMMENTS selection M+H, M-H. The data was normalized by the constant mean algorithm. The MS:MS_COMMENTS metabolites identifications and pathway analysis nodes used the default features MS:MS_COMMENTS in this metabolomics workflow. MS:ION_MODE NEGATIVE MS:MS_RESULTS_FILE ST002738_AN004441_Results.txt UNITS:area Has m/z:Yes Has RT:Yes RT units:Minutes #END