#METABOLOMICS WORKBENCH KathrynWolhuter_20250909_231619 DATATRACK_ID:6409 STUDY_ID:ST004197 ANALYSIS_ID:AN006976 PROJECT_ID:PR002646 VERSION 1 CREATED_ON September 17, 2025, 9:44 am #PROJECT PR:PROJECT_TITLE Metabolomic analysis of PHACTR1 overexpressing and knockdown HT1080 stable cell PR:PROJECT_TITLE lines PR:PROJECT_SUMMARY Genetic studies have consistently linked the PHACTR1 gene to a range of vascular PR:PROJECT_SUMMARY and neurological diseases, underscoring its pivotal role in biology. However, PR:PROJECT_SUMMARY the full spectrum of PHACTR1-mediated signaling pathways remains largely PR:PROJECT_SUMMARY unexplored. To bridge this gap, we employed a multi-omic (transcriptomics, PR:PROJECT_SUMMARY proteomics, untargeted metabolomics, targeted lipidomics) integration pipeline PR:PROJECT_SUMMARY leveraging a toolbox of bioinformatic techniques to identify disruptions in PR:PROJECT_SUMMARY biologically relevant pathways. By generating and integrating transcriptomic, PR:PROJECT_SUMMARY proteomic, metabolomic, and lipidomic datasets from HT1080 cells with PHACTR1 PR:PROJECT_SUMMARY overexpression or knockdown, we reveal the broad scope of PHACTR1 signaling. PR:PROJECT_SUMMARY This integrated approach has uncovered new roles for PHACTR1 in regulating the PR:PROJECT_SUMMARY cell cycle, iron homeostasis, and mitochondrial biogenesis and bioenergetics. PR:PROJECT_SUMMARY Our findings deepen our understanding of PHACTR1’s functions and showcase the PR:PROJECT_SUMMARY power of multi-omics approaches to dissect complex disease mechanisms. PR:INSTITUTE Victor Chang Cardiac Research Institute PR:DEPARTMENT Vascular Biology PR:LAST_NAME Wolhuter PR:FIRST_NAME Kathryn PR:ADDRESS Lowy Packer Building, Sydney, NSW, 2010, Australia PR:EMAIL k.wolhuter@victorchang.edu.au PR:PHONE +61 (02) 9295 8625 #STUDY ST:STUDY_TITLE Untargeted metabolomics on PHACTR1 overexpressing and knockdown HT1080 stable ST:STUDY_TITLE cell lines ST:STUDY_SUMMARY The PHACTR1 gene is involved in a range of vascular and neurological diseases, ST:STUDY_SUMMARY underscoring its pivotal role in biology. As part of a larger multi-omics study, ST:STUDY_SUMMARY we performed untargeted metabolomics (HILIC and XBD coupled with LC-MS) on ST:STUDY_SUMMARY stable HT1080 cell lines with overexpression or knockdown of PHACTR1, comparing ST:STUDY_SUMMARY them to scrambled control cells, to understand how PHACTR1 expression affects ST:STUDY_SUMMARY cellular metabolites. Cell lines were generated by lentiviral transfection with ST:STUDY_SUMMARY viral particles produced in Lenti-X 293T cells. Stable cell lines were selected ST:STUDY_SUMMARY with puromycin and maintained in media containing the selection agent. ST:INSTITUTE Victor Chang Cardiac Research Institute ST:DEPARTMENT Vascular Biology ST:LAST_NAME Wolhuter ST:FIRST_NAME Kathryn ST:ADDRESS Lowy Packer Building ST:EMAIL k.wolhuter@victorchang.edu.au ST:PHONE +61 (02) 9295 8625 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male SU:CELL_BIOSOURCE_OR_SUPPLIER ATCC SU:CELL_STRAIN_DETAILS HT1080, RRID:CVCL_0317 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - KD_01 Sample source:HT1080 cells | Treatment:PHACTR1 knockdown | Injection order:1 RAW_FILE_NAME(Raw file name (1))=20240304_HILIC_1.mzML; RAW_FILE_NAME(Raw file name (2))=20240304_XDB_1.mzML SUBJECT_SAMPLE_FACTORS - KD_04 Sample source:HT1080 cells | Treatment:PHACTR1 knockdown | Injection order:4 RAW_FILE_NAME(Raw file name (1))=20240304_HILIC_4.mzML; RAW_FILE_NAME(Raw file name (2))=20240304_XDB_4.mzML SUBJECT_SAMPLE_FACTORS - KD_05 Sample source:HT1080 cells | Treatment:PHACTR1 knockdown | Injection order:5 RAW_FILE_NAME(Raw file name (1))=20240304_HILIC_5.mzML; RAW_FILE_NAME(Raw file name (2))=20240304_XDB_5.mzML SUBJECT_SAMPLE_FACTORS - KD_06 Sample source:HT1080 cells | Treatment:PHACTR1 knockdown | Injection order:6 RAW_FILE_NAME(Raw file name (1))=20240304_HILIC_6.mzML; RAW_FILE_NAME(Raw file name (2))=20240304_XDB_6.mzML SUBJECT_SAMPLE_FACTORS - KD_10 Sample source:HT1080 cells | Treatment:PHACTR1 knockdown | Injection order:10 RAW_FILE_NAME(Raw file name (1))=20240304_HILIC_10.mzML; RAW_FILE_NAME(Raw file name (2))=20240304_XDB_10.mzML SUBJECT_SAMPLE_FACTORS - KD_19 Sample source:HT1080 cells | Treatment:PHACTR1 knockdown | Injection order:19 RAW_FILE_NAME(Raw file name (1))=20240304_HILIC_19.mzML; RAW_FILE_NAME(Raw file name (2))=20240304_XDB_19.mzML SUBJECT_SAMPLE_FACTORS - S_09 Sample source:HT1080 cells | Treatment:scrambled control | Injection order:9 RAW_FILE_NAME(Raw file name (1))=20240304_HILIC_9.mzML; RAW_FILE_NAME(Raw file name (2))=20240304_XDB_9.mzML SUBJECT_SAMPLE_FACTORS - S_13 Sample source:HT1080 cells | Treatment:scrambled control | Injection order:13 RAW_FILE_NAME(Raw file name (1))=20240304_HILIC_13.mzML; RAW_FILE_NAME(Raw file name (2))=20240304_XDB_13.mzML SUBJECT_SAMPLE_FACTORS - S_14 Sample source:HT1080 cells | Treatment:scrambled control | Injection order:14 RAW_FILE_NAME(Raw file name (1))=20240304_HILIC_14.mzML; RAW_FILE_NAME(Raw file name (2))=20240304_XDB_14.mzML SUBJECT_SAMPLE_FACTORS - S_20 Sample source:HT1080 cells | Treatment:scrambled control | Injection order:20 RAW_FILE_NAME(Raw file name (1))=20240304_HILIC_20.mzML; RAW_FILE_NAME(Raw file name (2))=20240304_XDB_20.mzML SUBJECT_SAMPLE_FACTORS - S_25 Sample source:HT1080 cells | Treatment:scrambled control | Injection order:25 RAW_FILE_NAME(Raw file name (1))=20240304_HILIC_25.mzML; RAW_FILE_NAME(Raw file name (2))=20240304_XDB_25.mzML SUBJECT_SAMPLE_FACTORS - S_27 Sample source:HT1080 cells | Treatment:scrambled control | Injection order:27 RAW_FILE_NAME(Raw file name (1))=20240304_HILIC_27.mzML; RAW_FILE_NAME(Raw file name (2))=20240304_XDB_27.mzML SUBJECT_SAMPLE_FACTORS - OE_03 Sample source:HT1080 cells | Treatment:PHACTR1 overexpressor | Injection order:3 RAW_FILE_NAME(Raw file name (1))=20240304_HILIC_3.mzML; RAW_FILE_NAME(Raw file name (2))=20240304_XDB_3.mzML SUBJECT_SAMPLE_FACTORS - OE_07 Sample source:HT1080 cells | Treatment:PHACTR1 overexpressor | Injection order:7 RAW_FILE_NAME(Raw file name (1))=20240304_HILIC_7.mzML; RAW_FILE_NAME(Raw file name (2))=20240304_XDB_7.mzML SUBJECT_SAMPLE_FACTORS - OE_15 Sample source:HT1080 cells | Treatment:PHACTR1 overexpressor | Injection order:15 RAW_FILE_NAME(Raw file name (1))=20240304_HILIC_15.mzML; RAW_FILE_NAME(Raw file name (2))=20240304_XDB_15.mzML SUBJECT_SAMPLE_FACTORS - OE_17 Sample source:HT1080 cells | Treatment:PHACTR1 overexpressor | Injection order:17 RAW_FILE_NAME(Raw file name (1))=20240304_HILIC_17.mzML; RAW_FILE_NAME(Raw file name (2))=20240304_XDB_17.mzML SUBJECT_SAMPLE_FACTORS - OE_22 Sample source:HT1080 cells | Treatment:PHACTR1 overexpressor | Injection order:22 RAW_FILE_NAME(Raw file name (1))=20240304_HILIC_22.mzML; RAW_FILE_NAME(Raw file name (2))=20240304_XDB_22.mzML SUBJECT_SAMPLE_FACTORS - OE_23 Sample source:HT1080 cells | Treatment:PHACTR1 overexpressor | Injection order:23 RAW_FILE_NAME(Raw file name (1))=20240304_HILIC_23.mzML; RAW_FILE_NAME(Raw file name (2))=20240304_XDB_23.mzML #COLLECTION CO:COLLECTION_SUMMARY All cell samples (n=6/cell line) were generated in parallel using the same CO:COLLECTION_SUMMARY passage cells, tissue culture reagents and culture conditions followed by CO:COLLECTION_SUMMARY concurrent harvesting. Cells were plated 24 hours prior to harvest at 2.2 x 105 CO:COLLECTION_SUMMARY cells/mL in DMEM with 10% FBS and 0.2 μg/mL puromycin for stable cell lines. To CO:COLLECTION_SUMMARY harvest, plates were washed once with ice-cold PBS and transferred to ice for CO:COLLECTION_SUMMARY metabolite extraction. CO:SAMPLE_TYPE Cultured cells CO:STORAGE_CONDITIONS -80℃ CO:COLLECTION_TUBE_TEMP 4℃ #TREATMENT TR:TREATMENT_SUMMARY HT1080 stable cell lines with PHACTR1 gene overexpression or knockdown and TR:TREATMENT_SUMMARY scrambled controls were made by lentiviral transfection. The PHACTR1 TR:TREATMENT_SUMMARY overexpression (OE) gene construct (NM_030948.6) was cloned into the lentiviral TR:TREATMENT_SUMMARY expression vector (pLVX-EF1a-Ires-Puro, Takara Bio, #631988) and PHACTR1 siRNA TR:TREATMENT_SUMMARY knockdown (KD) construct (pSMART hEF1a with PHACTR1 siRNA, Horizon, TR:TREATMENT_SUMMARY #V3SH11240-225060755), along with the empty vector control and scrambled siRNA TR:TREATMENT_SUMMARY control (pSMART hEF1a with non-targeting control, Horizon, #DHA-VSC11) plasmids, TR:TREATMENT_SUMMARY were transfected into Lenti-X 293T cells (Takara, # Cat#632180) using the TR:TREATMENT_SUMMARY Lenti-X Packaging Single Shots system (Takara, #631275). Transfected cells were TR:TREATMENT_SUMMARY cultured in DMEM with 10% Tet System Approved FBS (Takara, # 631106) for 48 TR:TREATMENT_SUMMARY hours. Cell culture media containing lentiviral particles for each plasmid were TR:TREATMENT_SUMMARY harvested and combined with 8 µg/mL polybrene before addition to HT1080 cells. TR:TREATMENT_SUMMARY Cells were incubated at 32°C, 5% CO2 for 6 hours followed by 72 hours at 37°C, TR:TREATMENT_SUMMARY 5% CO2. Transduced cells were selected by the addition of 0.4 μg/mL puromycin TR:TREATMENT_SUMMARY for 7 days. Stably transduced cell lines were maintained in complete DMEM with TR:TREATMENT_SUMMARY the addition of 0.2 μg/mL puromycin. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Aqueous metabolites were extracted using Agilent’s biphasic MeOH:CHCl3:H2O SP:SAMPLEPREP_SUMMARY (2:2:1.8) extraction protocol (Agilent Technologies application note 5989-7407). SP:SAMPLEPREP_SUMMARY In brief, cells were washed once with ice-cold PBS before quenching with 300 µl SP:SAMPLEPREP_SUMMARY 2:1 ice-cold MeOH:H2O on dry ice. Plates were transferred to wet ice to scrape SP:SAMPLEPREP_SUMMARY cells. Collected cell pellets were moved to pre-cooled 2 mL Eppendorf and plates SP:SAMPLEPREP_SUMMARY were washed with 300 µL ice-cold MeOH:H2O. The wash was collected and added to SP:SAMPLEPREP_SUMMARY cell pellets. Samples and, a blank extract containing 600 µL MeOH:H2O, were SP:SAMPLEPREP_SUMMARY kept on wet ice throughout extraction by the addition of 240 µl ice-cold CHCl3 SP:SAMPLEPREP_SUMMARY spiked with a mixture of internal standards containing L-valine-13C5,15N (Sigma SP:SAMPLEPREP_SUMMARY Aldrich, #600148) and unlabeled caffeine (Sigma Aldrich, #1000949613). Samples SP:SAMPLEPREP_SUMMARY were vortexed then 240 µl ice-cold H2O was added with vortexing followed by 240 SP:SAMPLEPREP_SUMMARY µl ice-cold CHCl3. Samples were vortexed 3 x 10 sec then centrifuged at 5,000 SP:SAMPLEPREP_SUMMARY x g for 40 min at 4°C. The aqueous phase was transferred to a fresh Eppendorf SP:SAMPLEPREP_SUMMARY tube and extracts were lyophilized at room temperature and stored at -80°C. SP:SAMPLEPREP_SUMMARY Prior to analysis, samples were reconstituted in 90 µl MeOH:ACN:H2O (1:1:1.6). SP:SAMPLEPREP_SUMMARY Samples were vortexed and sonicated in a water bath for 20 min at room SP:SAMPLEPREP_SUMMARY temperature. Samples were then centrifuged (21,000xg, 10 min, 4°C) and SP:SAMPLEPREP_SUMMARY transferred into sample vials with glass inserts for analysis. SP:EXTRACTION_METHOD Agilent’s biphasic MeOH:CHCl3:H2O (2:2:1.8) extraction protocol (Agilent SP:EXTRACTION_METHOD Technologies application note 5989-7407) SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION MeOH:ACN:H2O (1:1:1.6) SP:SAMPLE_SPIKING L-valine-13C5,15N (Sigma Aldrich, #600148) and unlabeled caffeine (Sigma SP:SAMPLE_SPIKING Aldrich, #1000949613) #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Extracted cellular metabolites were analyzed in one batch along with technical CH:CHROMATOGRAPHY_SUMMARY quality control (TQC) samples spaced 1 in every 10. Samples were analyzed by MS1 CH:CHROMATOGRAPHY_SUMMARY followed by 5 iterative QC (iQC) samples at the end of the worklist analyzed by CH:CHROMATOGRAPHY_SUMMARY MS2. Samples (5 µl) were separated on a 1290 Infinity II Bio High-Speed Pump CH:CHROMATOGRAPHY_SUMMARY and Infinity II Bio Multisampler HPLC system (Agilent). Samples were separated CH:CHROMATOGRAPHY_SUMMARY on an ZORBAX Eclipse XDB-C18 (2.1 x 100 mm, 1.8 µm, Agilent) with UHPLC Guard CH:CHROMATOGRAPHY_SUMMARY ZORBAX (2.1 x 5 mm, 1.8 µm, Agilent). The gradients were as follows: starting CH:CHROMATOGRAPHY_SUMMARY at 0% B for 2 min followed by an increase of solvent to 50% B over 15 min then CH:CHROMATOGRAPHY_SUMMARY 95% B over 2 min. The solvent was held at 95% B for 5 min (total 22 min). CH:CHROMATOGRAPHY_SUMMARY Equilibration was as follows: the solvent was decreased from 95% B to 0% B over CH:CHROMATOGRAPHY_SUMMARY 0.5 min. This was followed by a 4 min post run time (total 26.5 min sample CH:CHROMATOGRAPHY_SUMMARY cycle/sample). CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Agilent ZORBAX Eclipse XDB-C18 (100 x 2.1 mm, 1.8 µm) CH:SOLVENT_A 100% Water; 10mM ammonium formate; 0.1% Formic acid CH:SOLVENT_B 10% Water/90% Acetonitrile; 0.1% Formic acid; 10mM ammonium formate CH:FLOW_GRADIENT 0-2 min: 0% B - 0% B; 2-17 min: 0% B - 50% B; 17-19 min: 50% B - 95% B; 19-22 CH:FLOW_GRADIENT min: 95% B - 95% B; 22-22.5 min: 95% B - 0% B; 22.5-26.5 min: 0% B - 0% B CH:FLOW_RATE 0.25 mL/min CH:COLUMN_TEMPERATURE 35°C #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Innovation Centre’s Freedman Foundation Metabolomics Facility AN:OPERATOR_NAME Kathryn Wolhuter #MS MS:INSTRUMENT_NAME Agilent 6456 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The following mass spectrometer conditions were used; gas temperature 200°C, MS:MS_COMMENTS gas flow rate 10 L/min, nebulizer 35 psi, Sheath gas temperature 300°C, MS:MS_COMMENTS capillary voltage 3500 V and sheath gas flow 12 L/min. MS1 data was acquired for MS:MS_COMMENTS m/z range 100-1200 at a scan rate for 3 spectra/sec. Iterative MS2 acquisitions MS:MS_COMMENTS were performed using: MS1 m/s range 100-1200, MS1 scan rate 5 spectra/sec, MS2 MS:MS_COMMENTS m/z range 50-1600, MS2 scan rate 10 spectra/sec, isolate width 1.3 amu, MS:MS_COMMENTS accumulation target value 25000 ions, active exclusion after 2 spectra for 0.2 MS:MS_COMMENTS min with a mass tolerance of 20 ppm. MS1 TQCs were monitored for changes in peak MS:MS_COMMENTS area, width, and retention time to determine the performance of the LC-MS MS:MS_COMMENTS analysis. Feature picking and identification from MS1 spectral data was MS:MS_COMMENTS performed using MassHunter Explorer 1 (Agilent). Feature identification MS:MS_COMMENTS settings: Ion height filter (counts): 600, Ion Species: +H, +Na, +NH4, RT MS:MS_COMMENTS correction against TQC, Max time shift between samples: 0.5min + 0.5%, RT MS:MS_COMMENTS tolerance: 0.3min, Mass tolerance: 20ppm. Features were Log2 transformed and MS:MS_COMMENTS filtered (80% frequency, <=25% coefficient of variation in at least one group). MS:MS_COMMENTS Features were identified against MassHunter METLIN Metabolite accurate mass MS:MS_COMMENTS Personal Compound Database and Library with mass tolerance of 5 ppm. MS:MS_COMMENTS Significantly changed features were identified with Benjamini-Hochberg FDR<0.1 MS:MS_COMMENTS against scrm ctrl. For MS2 validation of significantly changed features, iTQC MS:MS_COMMENTS spectral data was analyzed in MassHunter Qualitative Analysis (Agilent) using MS:MS_COMMENTS Find by Molecular Feature algorithm against MassHunter METLIN accurate mass MS:MS_COMMENTS Personal Compound Database and Library. Spectral library search settings: min MS:MS_COMMENTS m/z: 30, min match score: 50, precursor tolerance 20±0.05 ppm, MS/MS peak MS:MS_COMMENTS height (counts) ≥ 10, limit to the largest 100, isotope spacing tolerance MS:MS_COMMENTS 0.0025 m.z/7 ppm, isotope model: common organic molecules. Additional Molecular MS:MS_COMMENTS Feature database search settings: Use peaks with height ≥ 600, Modifiers: +H, MS:MS_COMMENTS +Na, +NH4, Mass match tolerance: 2.00 mDa, Extract separate MS/MS spectrum per MS:MS_COMMENTS collision energy: True. MS/MS spectral data for all five iTQC was exported as a MS:MS_COMMENTS single combined MGF. Significant features found in MassHunter Explorer were then MS:MS_COMMENTS manually validated against the combined MGF using SIRIUS 5.8.6 by Molecular MS:MS_COMMENTS Formular Identification, CSI:FingerID Fingerprint Prediction and Structural MS:MS_COMMENTS Database Searching against HMDB and KEGG.64 Only MS2-validated human metabolites MS:MS_COMMENTS with HMDB and/or KEGG IDs were used for pathway analysis. MS:MS_RESULTS_FILE ST004197_AN006976_Results.txt UNITS:ion intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END