Metabolome analyses of oncogene-induced senescent IMR-90 cells

Methods

Cell culture

IMR-90 ER:Ras (H-RasG12V) cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 200 mM L-glutamine, 1 mM sodium pyruvate, 10% (v/v) heat-inactivated fetal bovine serum (FBS), and penicillin/streptomycin (P/S). To induce quiescence, IMR-90 ER:Ras cells were cultured in the above medium containing 0.1% FBS for 2 days. For oncogene-induced senescence, IMR-90 ER:Ras cells were treated with 100 nM 4-hydroxytamoxifen (4-OHT) for 6 days.

Metabolite extraction

Culture medium was aspirated from the dishes and the cells were washed twice with 5% mannitol solution (10 mL and 2 mL for the first and second washes, respectively). The cells were then treated with 800 µL of methanol and incubated at room temperature for 30 sec to suppress enzyme activity. Next, 550 µL of Milli-Q water containing internal standards (H3304-1002, Human Metabolome Technologies, Inc. (HMT), Tsuruoka, Yamagata, Japan) was added to the cell extract, followed by further incubation at room temperature for 30 sec. The cell extract was then centrifuged at 2,300 × g and 4ºC for 5 min, after which 700 µL of the supernatant was centrifugally filtered through a Millipore 5-kDa cut-off filter (UltrafreeMC-PLHCC, HMT) at 9,100 × g and 4ºC for 120 min to remove macromolecules. Subsequently, the filtrate was evaporated to dryness under vacuum and reconstituted in 50 µL of Milli-Q water for metabolome analysis at HMT.

Metabolome analysis

Metabolome analysis was conducted using HMT’s Basic Scan package, capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), and the previously described method 1. Briefly, CE-TOFMS analysis was carried out using an Agilent CE capillary electrophoresis system equipped with an Agilent 6210 time-of-flight mass spectrometer (Agilent Technologies, Inc., Santa Clara, CA, USA). The systems were controlled by Agilent G2201AA ChemStation software version B.03.01 (Agilent Technologies) and connected by a fused silica capillary (50 μm internal diameter × 80 cm total length) containing commercial electrophoresis buffer (H3301-1001 and I3302-1023 for cation and anion analyses, respectively, HMT) as the electrolyte. The spectrometer scanned from m/z 50 to 1,000 and peaks were identified using MasterHands automatic integration software (Keio University, Tsuruoka, Yamagata, Japan) in order to obtain peak information, including m/z, peak area, and migration time (MT) 2. Signal peaks corresponding to isotopomers, adduct ions, and other product ions corresponding to known metabolites were excluded, and the remaining peaks were annotated according to HMT’s metabolite database, based on their m/z values and MTs. The areas of the annotated peaks were then normalized to those for internal standards and the amounts of the samples in order to obtain the relative quantity for each metabolite. The absolute quantities of 110 primary metabolites were quantified on the basis of one-point calibrations using their respective standards.

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