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MB Sample ID: SA196513

Local Sample ID:	RSL3_5
Subject ID:	SU002164
Subject Type:	Cultured cells
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116
Cell Biosource Or Supplier:	American Type Culture Collection (ATCC)
Cell Strain Details:	H9c2(2-1) is a subclone of the original clonal cell line derived from embryonic BD1X rat heart tissue by B. Kimes and B. Brandt and exhibits many of the properties of skeletal muscle.
Cell Passage Number:	3-10
Cell Counts:	80-90% confluency

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Subject:

Subject ID:	SU002164
Subject Type:	Cultured cells
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116
Cell Biosource Or Supplier:	American Type Culture Collection (ATCC)
Cell Strain Details:	H9c2(2-1) is a subclone of the original clonal cell line derived from embryonic BD1X rat heart tissue by B. Kimes and B. Brandt and exhibits many of the properties of skeletal muscle.
Cell Passage Number:	3-10
Cell Counts:	80-90% confluency

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
RSL3_5	SA196513	FL023973	RSL3	Treatment

Collection:

Collection ID:	CO002157
Collection Summary:	H9c2 cardioblasts were cultured according to the manufacturer’s recommendations (ATCC, Manassas, VA, USA) with minor modifications. Briefly, the cells were incubated in DMEM based modified media containing 4 mM L-glutamine, 4.5 g/L glucose, 1 mM sodium pyruvate, and 1.5 g/L sodium bicarbonate, pH 7.4, and supplemented with 10% fetal bovine serum and 1% antibiotic solution (HyClone, Logan, UT, USA) in a CO2 incubator containing 95% air and 5% CO2 at 37 ◦C. Cells with 80–90% confluence from passages 3–10 were used for experiments. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO). H9c2 cardioblasts demonstrate similar to the primary cardiomyocytes hypertrophic response as well as mitochondrial metabolism and morphology.
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR002176
Treatment Summary:	Cultured H9c2 cells reaching 80–90% confluence from passages 3–10 were used for experiments. Ferroptosis was induced by incubating the cells with 0.5 µM RSL3 for 3 h. In addition, the cells were exposed to RSL3 in the presence of 1 µM Fer-1, 0.2 µM XJB, and 0.6 µM TSM. Thus, experiments were performed in the following 8 groups: (i) control (n = 5), (ii) RSL3 (n = 6), (iii) Fer-1 (n = 6), (iv) XJB (n = 5), (v) TSM (n = 5), (vi) RSL3 + Fer-1 (n = 6), (vii) RSL3 + XJB (n = 6), and (viii) RSL3 + TSM (n = 5). Fer-1, XJB, or TSM were added to the culture medium simultaneously with RSL3. At the end of the incubation, the cells were harvested and processed for analysis by GC-MS.
Cell Pct Confluence:	80-90%

Sample Preparation:

Sampleprep ID:	SP002170
Sampleprep Summary:	Metabolites were extracted as previously described using 1 mL of cold MeOH/H2O (85:15). Samples were sonicated for 15 s (3×) on ice and centrifuged at 1400 rpm × 10 min at 4 ◦C (Rotor model: Eppendorf, FA-45-30-11). For protein quantification, the pellets were dried for 15 min in a rotary vacuum evaporator, then resuspended in 60 µL of denaturation buffer and sonicated for 1 min. All samples were centrifuged for 10 min at the maximum speed and the supernatant was used for protein quantification using the Bradford method. Supernatants were collected and evaporated to dryness using a SpeedVac (Savant AS160, Farmingdale, NY, USA). The metabolite samples were first derivatized through methoxyamination by adding 30 µL of 20 mg/mL solution of methoxyamine hydrochloride Antioxidants 2022, 11, 278 4 of 16 in pyridine (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37 ◦C for 2 h. Afterward, trimethylsilylation was performed by adding 30 µL of N-tert-butyldimethylsilyl-Nmethyltrifluoroacetamide (MTBSTFA + 1% TBDMSCl; Sigma-Aldrich, St. Louis, MO, USA) and incubated for 1 h at 65 ◦C. The reaction mixture was centrifuged at 1300 rpm × 10 min at RT. Supernatants were transferred to analytical vials. Then, 20 µL per sample was added to glass vials with inserts followed by the addition of 1 mM 2-fluobiphenyl (Sigma-Aldrich, St. Louis, MO, USA) as an internal standard. Samples were processed by GC-MS-QP2010 (Shimadzu Scientific Instruments, Inc., Columbia, MD) using analytical conditions as previously described.

Sampleprep ID:

SP002170

Sampleprep Summary:

Metabolites were extracted as previously described using 1 mL of cold MeOH/H2O (85:15). Samples were sonicated for 15 s (3×) on ice and centrifuged at 1400 rpm × 10 min at 4 ◦C (Rotor model: Eppendorf, FA-45-30-11). For protein quantification, the pellets were dried for 15 min in a rotary vacuum evaporator, then resuspended in 60 µL of denaturation buffer and sonicated for 1 min. All samples were centrifuged for 10 min at the maximum speed and the supernatant was used for protein quantification using the Bradford method. Supernatants were collected and evaporated to dryness using a SpeedVac (Savant AS160, Farmingdale, NY, USA). The metabolite samples were first derivatized through methoxyamination by adding 30 µL of 20 mg/mL solution of methoxyamine hydrochloride Antioxidants 2022, 11, 278 4 of 16 in pyridine (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37 ◦C for 2 h. Afterward, trimethylsilylation was performed by adding 30 µL of N-tert-butyldimethylsilyl-Nmethyltrifluoroacetamide (MTBSTFA + 1% TBDMSCl; Sigma-Aldrich, St. Louis, MO, USA) and incubated for 1 h at 65 ◦C. The reaction mixture was centrifuged at 1300 rpm × 10 min at RT. Supernatants were transferred to analytical vials. Then, 20 µL per sample was added to glass vials with inserts followed by the addition of 1 mM 2-fluobiphenyl (Sigma-Aldrich, St. Louis, MO, USA) as an internal standard. Samples were processed by GC-MS-QP2010 (Shimadzu Scientific Instruments, Inc., Columbia, MD) using analytical conditions as previously described.

Combined analysis:

Analysis ID	AN003394
Analysis type	MS
Chromatography type	GC
Chromatography system	Shimadzu GCMS-QP2010 ultra
Column	Shimadzu SH-RXI (30m x 0.25mm,0.25um)
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Shimadzu QP2010 Ultra
Ion Mode	POSITIVE
Units	mM

Chromatography:

Chromatography ID:	CH002509
Instrument Name:	Shimadzu GCMS-QP2010 ultra
Column Name:	Shimadzu SH-RXI (30m x 0.25mm,0.25um)
Chromatography Type:	GC

MS:

MS ID:	MS003161
Analysis ID:	AN003394
Instrument Name:	Shimadzu QP2010 Ultra
Instrument Type:	Single quadrupole
MS Type:	EI
MS Comments:	Raw chromatography data were obtained and processed in GC-MS Labsolution Postrun Analysis software (Shimadzu Scientific Instruments, Inc., Columbia, MD) for identification of metabolites from their electron impact mass spectra by comparison to the database (NIST14/2014/EPA/NIH) [26]. Peak integration for all metabolites and multiple searches in the mass spectral library database resulted in a final database of 52 metabolic features chosen for this analysis. To assess analytical accuracy and precision, an external quality evaluation was performed using 2-fluorobiphenyl spiked into derivatization blank samples before running on the GC-MS. The quantitative analysis corresponding to the metabolite concentrations in each sample was calculated based on the internal standard in mM. A table in the format of comma-delimited (* csv) was created and uploaded to MetaboAnalyst.ca.
Ion Mode:	POSITIVE