Summary of Study ST003960
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002482. The data can be accessed directly via it's Project DOI: 10.21228/M8TC38 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST003960 |
| Study Title | Relative quantification of lactic acid secreted in media from cell lines treated with or without Antimycin A. |
| Study Summary | This study aimed to investigate the lactate secretion as a measurement of glycolytic switch in subjected cell lines during OXPHOS dysfunction using targeted GC-MS analysis. To determine the ROS-mediated metabolic shift toward glycolysis, we quantified the relative abundance of lactic acid in the growth media normalized by cell number from control HEK293T cells without Antimycin A treatment, and Antimycin A treated SDHAF2KO cells, using GC-MS and an MRM quantification strategy. Control HEK293T cells treated with Antimycin A showed significantly higher levels of lactic acid compared to untreated cells. However, media lactic acid was not increased in Antimycin A-treated SDHAF2KO cells, suggesting an active ROS-mediated glycolytic switch only in the presence of SDHAF2 during OXPHOS impairment. |
| Institute | University of Melbourne |
| Last Name | Roopasingam |
| First Name | Kugapreethan |
| Address | 30 Flemington Rd, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia. |
| k.roopasingam@unimelb.edu.au | |
| Phone | 0434297212 |
| Submit Date | 2025-05-29 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | gqd |
| Analysis Type Detail | GC-MS |
| Release Date | 2025-06-11 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002482 |
| Project DOI: | doi: 10.21228/M8TC38 |
| Project Title: | Complex II assembly drives metabolic adaptation to OXPHOS dysfunction. |
| Project Summary: | During acute oxidative phosphorylation (OXPHOS) dysfunction, the reverse activity of succinate dehydrogenase (Complex II) maintains the redox state of Coenzyme Q by utilizing either fumarate or oxygen as terminal electron acceptors. The tendency for one over another has been suggested to be tissue-specific, but the underlying mechanism and consequence of this is unknown. Using quantitative proteomics to screen a panel of HEK293T knockout cell lines, we identified an increase in SDHAF2 protein, a Complex II assembly factor that enhances the flavination of catalytic subunit SDHA, as critical for metabolic adaptation during OXPHOS stress in HEK293T cells. Loss of SDHAF2 during Complex III inhibition resulted in a reduction in Complex II F-site derived reactive oxygen species (ROS), a severe growth impairment, and a net reductive TCA cycle driven by an inability of mitochondria to support additional Complex II assembly. This in turn leads to use of fumarate as terminal electron acceptor at the cost of a ROS-mediated switch to glycolysis. Cell lines adapted to glycolysis did not accumulate SDHAF2 upon OXPHOS stress and exhibited a net reductive TCA cycle and mild growth phenotypes with or without SDHAF2 being present. Thus, our study reveals how Complex II assembly controls a balance between protection of the Q-pool and ROS-meditated signaling during oxidative stress in cells reliant on mitochondrial OXPHOS. |
| Institute: | University of Melbourne |
| Last Name: | Roopasingam |
| First Name: | Kugapreethan |
| Address: | 30 Flemington Rd, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia. |
| Email: | k.roopasingam@unimelb.edu.au |
| Phone: | 0434297212 |
Subject:
| Subject ID: | SU004097 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Treatment |
|---|---|---|---|
| SA452767 | 1-1_Fresh Media | 1-1_Fresh Media_001 | Control neg |
| SA452768 | 1-2_Fresh Media | 1-1_Fresh Media_002 | Control neg |
| SA452769 | 1-3_Fresh Media | 1-1_Fresh Media_003 | Control neg |
| SA452770 | 2-1_HEKWT | 2-1_HEKWT_001 | Control |
| SA452771 | 2-2_HEKWT | 2-1_HEKWT_002 | Control |
| SA452772 | 2-3_HEKWT | 2-1_HEKWT_003 | Control |
| SA452773 | 3-1_HEKWT_A-A | 3-1_HEKWT_A-A_001 | Treatment |
| SA452774 | 3-2_HEKWT_A-A | 3-1_HEKWT_A-A_002 | Treatment |
| SA452775 | 3-3_HEKWT_A-A | 3-1_HEKWT_A-A_003 | Treatment |
| SA452776 | 4-1_HEKSDHAF2_A-A | 4-1_HEKSDHAF2_A-A_001 | Treatment |
| SA452777 | 4-2_HEKSDHAF2_A-A | 4-1_HEKSDHAF2_A-A_002 | Treatment |
| SA452778 | 4-3_HEKSDHAF2_A-A | 4-1_HEKSDHAF2_A-A_003 | Treatment |
| Showing results 1 to 12 of 12 |
Collection:
| Collection ID: | CO004090 |
| Collection Summary: | Cells were seeded at 50% confluency in 6-well dishes containing DMEM media (Thermo Scientific) 24 hours prior to the experiment. For metabolite extraction, 50 ul of media was collected from wells with and without the cells. |
| Sample Type: | Media |
Treatment:
| Treatment ID: | TR004106 |
| Treatment Summary: | The drug treatment was performed for 8 hours prior to media collection and the collected media was snap-frozen before polar metabolite extraction. |
Sample Preparation:
| Sampleprep ID: | SP004103 |
| Sampleprep Summary: | A monophasic extraction protocol was used to extract lactic acid from the media. To 30 µL of media, 90 µL of 100% MeOH containing 1.2 nmol of 13C515N valine and 1.2 nmol of 13C6 sorbitol was added. Each sample was vortexed and then incubated at 4˚C for 10 min with continuous agitation (12 g) using an Eppendorf Thermomixer C. The samples were centrifuged at 4˚C for 10 min at 16000 g using an Eppendorf centrifuge 5430 R. The supernatant was transferred into a fresh 1.5 mL Eppendorf tube and the precipitate was discarded. A 10 µL aliquot of each sample was pooled to create the pooled biological quality control (PBQC). Ten µL of each study sample and the PBQC were transferred into HPLC inserts and evaporated at 30˚C to complete dryness, using a CHRIST RVC 2-33 CD plus speed vacuum, prior to the GC-MS analysis. |
Chromatography:
| Chromatography ID: | CH004943 |
| Instrument Name: | Shimadzu GC-2010 |
| Column Name: | Agilent DB-5 GC column (30 m x 0.25 mm, 1 µm) |
| Column Temperature: | 100℃ |
| Flow Gradient: | N/A |
| Flow Rate: | 1 mL/min |
| Solvent A: | N/A |
| Solvent B: | N/A |
| Chromatography Type: | GC |
Analysis:
| Analysis ID: | AN006508 |
| Analysis Type: | MS |
| Chromatography ID: | CH004943 |
| Num Factors: | 12 |
| Num Metabolites: | 3 |
| Units: | Ratio of Relative abundance |
