Re 80

Saos-2 cells (0.5×10⁶) were seeded in triplicate wells of 6-well plates in high glucose DMEM supplemented with supplemented with 10% fetal bovine serum (FBS), 100μg/ml penicillin and100 μg/ml streptomycin (Gibco, Invitrogen) and 2mM L-glutamine. Duplicate plates were seeded for cell counts. After 24 h from doxycyline supplementation to the medium (2.5μg/ml), cells were washed with PBS and received heavy ¹³C-glucose or ¹³C¹⁵N-glutamine-substituted media (Cambridge Isotopes).
Extraction of metabolites was performed at 1, 6, 12 and 24h upon supplementation of heavy labeled media (0h = 24h from TAp63α induction via doxycycline supplementation). Cells were washed with PBS and metabolites extracted using methanol/acetonitrile/dH2O (5:3:2) (1–2×10⁶ cells per ml). Samples were vortexed for 30minutes at 4°C and thus centrifuged at 16000g for 15min at 4°C. Supernatants were collected for subsequent analyses. MS analyses were performed in polarity switch mode on an Orbitrap Exactive (Thermo Scientific) in line with an Accela autosampler and an Accela 600 pump (Thermo Scientific). Column hardware consisted of a Sequant ZIC-pHILIC column (2.1×150mm, 5μm) coupled to a Sequant ZIC-pHILIC guard column (2.1×20mm, 5 μm) (Merck). Flow rate was 100 ml min⁻¹, buffers consisted of acetonitrile (ACN) for A, and 20mM (NH₄)₂CO₃, 0.1% NH₄OH in ddH₂O for B.
Gradient ran from 80% to 40% ACN in 20min, followed by a wash at 20% ACN and re-equilibration at 80% ACN. Metabolites were identified and quantified using LCquan software (Thermo Scientific). Metabolites were positively identified on the basis of exact mass within 5 p.p.m., further validated by concordance with standard retention times and plotted as the peak area for each metabolite. Isotopomer distributions were determined on the basis of the expected reactions involving the investigated metabolite and by looking the actual isotopomeric distribution of mass spectra chromatograms. Results were graphed with Graphpad Prism 5.01 (Graphpad Software Inc) as means + SD. Statistical analyses were performed with the same software, as a result of paired t-test or two-way ANOVA among the results obtained from doxycycline supplemented (dox+) and non-supplemented (dox-) cells at the steady state or in a time-dependent fashion (independent variables being doxycycline supplementation and time-course assays).

i) Sample quenching, extraction, and preparation: All chemicals used for LC-MS metabolomic analyses were obtained from Sigma-Aldrich (Taufkirchen, Germany). Cells were collected by centrifugation at 8,000 × g for 8 min at room temperature (Eppendorf 5430R, Hamburg, Germany). The cell samples were quenched and extracted rapidly with 900 μL of 80:20 MeOH/H₂O (−80°C) and then frozen in liquid nitrogen. The samples were then frozen-thawed three times to release metabolites from the cells. The supernatant was collected after centrifugation at 15,000 × g for 5 min at −4°C and then stored at −80°C. The remaining cell pellets were re-suspended in 500 μL of 80:20 MeOH/H₂O (−80°C) and the above extraction process was repeated. The supernatant from the second extraction was pooled with that from the first extraction and stored at −80°C until LC-MS analysis [27 (link)]; ii) LC-MS analysis: The chromatographic separation was achieved with a SYnergi Hydro-RP (C18) 150 mm × 2.0 mm I.D., 4 μm 80 Å particles column (Phenomenex, Torrance, CA, USA) at 40°C. Mobile phase A (MPA) is an aqueous 10 mM tributylamine solution with pH 4.95 adjusted with acetic acid and Mobile phase B (MPB) is 100% methanol of HPLC grade (Darmstadt, Germany). The optimized gradient profile was determined as follows: 0 min (0% B), 8 min (35% B), 18 min (35% B), 24 min (90% B), 28 min (90% B), 30 min (50% B), 31 min (0% B). A 14-minute post-time equilibration was employed, bringing total run-time to 45 min. Flow rate was set as a constant 0.2 mL/min [57 (link)]. LC-MS analysis was conducted on an Agilent 1260 series binary HPLC system (Agilent Technologies, Waldbronn, Germany) coupled to an Agilent 6410 triple quadrupole mass analyser equipped with an electrospray ionization (ESI) source. Injected sample volume for all cases was 10 μL; capillary voltage was 4000 V; and nebulizer gas flow rate and pressure were 10 L/min and 50 psi, respectively. Nitrogen nebulizer gas temperature was 300°C. The MS was operated in negative mode for multiple reaction monitoring (MRM) development, method optimization, and sample analysis. Data were acquired using Agilent Mass Hunter workstation LC/QQQ acquisition software (version B.04.01) and chromatographic peaks were subsequently integrated via Agilent Qualitative Analysis software (version B.04.00); iii) Targeted metabolite analysis: a total of 24 metabolites were selected for LC-MS based targeted metabolite analysis in this study. The abbreviations, molecular weights and MRM values determined and optimized for each of the 24 detected metabolites as well as the product ion formulas were provided in Additional file 1: Table S1. The standard compounds for these 24 metabolites were purchased from Sigma, and their MS and MS/MS experimental parameters were optimized with the mix standard solution. All metabolomics profile data was first normalized by the internal control and the cell numbers of the samples, and then subjected to Principal Component Analysis using software SIMCA-P 11.5 [58 (link)].

Soluble sugars and hexose phosphates were extracted and derivatized according to Wang et al. [34] (link). Briefly, 0.1 g sample was extracted in 1.4 ml 75% methanol with ribitol added as internal standard. After fractionation of non-polar metabolites into chloroform, 2 and 100 µl of the polar phase of each sample were taken and transferred into 2.0 ml Eppendorf vials for highly abundant metabolites (such as Sor, Suc, Glc, and Fru) and less abundant metabolites (such as G6P and F6P), respectively. They were dried under vacuum without heating and then derivatized with methoxyamine hydrochloride and N-methyl-N-trimethylsilyl-trifluoroacetamide sequentially [35] (link). After derivatization, metabolites were analyzed with an Agilent 7890A GC/5975C MS (Agilent Technology, Palo Alto, CA, USA) [34] (link). Metabolites were identified by comparing fragmentation patterns with those in a mass spectral library generated on our GC/MS system and an annotated quadrupole GC–MS spectral library downloaded from the Golm Metabolome Database (http://csbdb.mpimp-golm. mpg.de/csbdb/gmd/msri/gmd_msri.html) and quantified based on standard curves generated for each metabolite and internal standard.
The tissue residue after 75% methanol extraction for GC-MS analysis was re-extracted with 80% (v/v) ethanol at 80°C three times, and the pellet was retained for determination of starch. After digesting the residue with 30 U of amyloglucosidase (EC 3.2.1.3) at pH 4.5 overnight, starch was determined enzymatically as glucose equivalents [36] (link).

Leaf carotenoids, chlorophylls, and tocopherols were extracted in 2 mL Eppendorf tubes from 4 mg of freeze-dried leaf tissue, using 375 µL of methanol as the extraction solvent and a 25 µL of 10% (w/v) solution of canthaxanthin in chloroform (Sigma) as the internal standard. Tissue was lysed by adding 4 mm glass beads and grinding for 1 min at 30 Hz in a TissueLyser II (QIAGEN, Venlo, Netherlands) and the extraction was carried out by adding 400 µL of Tris-NaCl pH 7.5 and 800 µL of chloroform. Thoroughly mixed samples were centrifuged for 5 min at 13,000 rpm at 4 °C and the organic phase was transferred into a new tube and evaporated using a SpeedVac system (Eppendorf Concentrator plus, Hamburg, Germany). The extracted metabolites were then completely redissolved in 200 µL of acetone, filtered with 0.2 µm filters into amber-colored 2 mL glass vials and a 10-µL aliquot of each sample was then injected onto an Agilent Technologies 1200 series HPLC system (Agilent Technologies, Santa Clara, CA, USA). A C30 reverse-phase column (YMC Carotenoid, 250 × 4.6 mm × 3 µm, YMC CO., Kyoto, Japan) was used, with three mobile phases consisting of methanol (solvent A), water/methanol (20/80 v/v) containing 0.2% ammonium acetate (w/v) (solvent B), and tert-methyl butyl ether (solvent C). Metabolites were separated following the following gradient: 95% A, 5% B isocratically for 12 min, a step-up to 80% A, 5% B, 15% C at 12 min, followed by a linear gradient up to 30% A, 5% B, and 65% C by 30 min. The flow rate was maintained at 1 mL/min. The HPLC equipment was coupled to a Photometric Diode Array (PDA) detector (Santa Clara, CA, USA) allowing the detection of the full UV-visible absorption spectra of the different metabolites. Peak areas of chlorophylls at 650 nm and carotenoids at 472 nm were determined using Agilent ChemStation software. A fluorescence detector at 330 nm was used for tocopherol identification. The quantification of the compounds of interest was done by using a concentration curve built with a commercial standard (Sigma-Aldrich, Steinheim, Germany) [51 (link)]. For the rest of isoprenoid metabolites, approximately 7 mg of freeze-dried tissue were mixed with 500 µL of THF:MeOH (Analytical grade, Normapur) 1:1 buffered with 10% of water (v/v), thoroughly mixed, centrifuged, transferred to an amber vial, and injected into a Waters Acquity UPLC™ (Milford, MA, USA) coupled to a Waters Synapt G2 MS QTOF equipped with an atmospheric pressure chemical ionization (APCI) source. Prenyllipids were separated on an Acquity BEH C18 column (50 × 2.1 mm, 1.7 µm) under the following conditions: Solvent A = water; Solvent B = MeOH; 80–100% B in 4.0 min, 100% B for 2.5 min, re-equilibration at 80% B for 2.0 min. The flow rate was 500 µL/min, and the injection volume was 2.5 µL. Standards of HPLC grade (≥99.5%) were purchased from Sigma-Aldrich. PQ-9 and PC-8 standards were purified in house [52 ].

For the analysis of the antioxidant activity, samples were prepared with a two-step extraction. The extraction procedure was developed based on a method described by Li et al. [72 (link)] and was used with the following modifications: approximately 500 mg freeze-dried powder was weighed into a tube containing 10 mL enzyme solution (0.4 mg lysozyme· mL⁻¹, Sørensen buffer; pH 7.4) and mixed for 45 min at 37 °C. The tube was centrifuged at 3226× g for 15 min at 15 °C. The residue was re-suspended with 80% ethanol (v/v), stirred for 1 min, and centrifuged under the same conditions as previously mentioned. Both supernatants were analyzed for total phenolic content (TPC), a well-known indicator of antioxidant activity, and Trolox equivalent antioxidant capacity (TEAC). The total amount was calculated as the sum of the two extracts.

The extraction of total flavonoids was carried out following the same procedure as for the extraction of phenols, although the sample was re-suspended in ethanol at 80% at a concentration of 1 mg/mL.
The total flavonoids were quantified following the method proposed by Chang et al. [82 (link)]. Firstly, 2 mL of ethanol were taken, to which we added 0.5 mL of extract (1 mg/mL re-suspended in 80% ethanol, 3 replicates) and 0.15 mL of NaNO₂ (1 M in ethanol). The mixture was stirred and left to settle for 3 min. Then, 0.15 mL of AlCl₃ (10%, in ethanol) was added, and the sample was stirred again for 3 min. Subsequently, we added 1 mL of NaOH (1 M in distilled water) and ethanol to 5 mL of total volume.
The solutions were mixed again after the last steps and kept in the dark for 40 min. Then, absorbance was measured at 415 nm using a UV-30 spectrophotometer. The blank was obtained by replacing the amount of diluted extract with ethanol.
The results are expressed in mg of quercetin equivalents (QE) per mg of dry weight.