L proline

143B‐TK⁻ osteosarcoma‐derived cybrid cells were grown in High‐Glucose (4.5 g/l) DMEM containing Sodium Pyruvate and GlutaMAX™ (Gibco‐Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Gibco‐Thermo Fisher Scientific) and 50 μg/ml uridine (Sigma‐Aldrich). Cells transduced with expression vectors containing a puromycin resistance cassette were grown in the presence of 1 μg/ml puromycin (InvivoGen). If it was hygromycin, the final concentration of the hygromycin B was 100 μg/ml (InvivoGen), and for neomycin‐resistant cells, the final concentration of geneticin was 500 μg/ml (Gibco‐Thermo Fisher Scientific).
The cells used in the SILAC experiments were grown in DMEM for SILAC (Gibco‐Thermo Fisher Scientific) plus 10% dialyzed serum (Gibco‐Thermo Fisher Scientific) and 50 μg/ml uridine, supplemented either with unlabeled L‐lysine monohydrochloride (K₀), L‐arginine (R₀), and L‐proline (“Light” conditions) or with L‐lysine‐¹³C₆,¹⁵N₂ hydrochloride (K₈), L‐arginine‐¹³C₆,¹⁵N₄ hydrochloride (R₁₀), and L‐proline (“Heavy” conditions), all from Sigma‐Aldrich.

A total of 1.7 g yeast basic nitrogen source (YNB without amino acids and ammonium sulfate, BD), 5 g ammonium sulfate (VETEC^TM, Merck), 20 g d-glucose, 0.1 g l-arginine, 0.1 g l-cysteine, 0.1 g l-lysine, 0.1 g l-threonine, 0.05 g l-aspartic acid, 0.05 g l-Isoleucine, 0.05 g l-phenylalanine, 0.05 g l-proline, 0.05 g l-serine, 0.05 g l-tyrosine, 0.05 g l-valine, 0.05 g l-methionine, 0.1 g l-tryptophan, 0.05 g l-histidine, 0.1 g l-uracil, 0.1 g l-leucine, 0.1 g l-adenine were added to 1 L deionized H₂O. All amino acids were purchased from Sigma-Aldrich. After autoclaving for 45 min, the SC medium was stored at room temperature. In all, 2% (m/v) glucose was supplemented to the medium before use.

Choline chloride (>98.0%), l-(+)-Lactic acid (>98.0%), glycerol (>99.5%), 1,2-propanediol (>99%) and 2,9-Dihydroxy-1,10-dimethoxyaporphine (boldine) analytical standard (purity ≥ 98%), were purchased from Sigma-Aldrich (Steinheim, Germany). Citric acid (>98%), levulinic acid (>98%), l-Proline (>99%), oxalic acid (>99%), sodium carbonate (>99.9%), gallic acid (>98.0%), and Folin–Ciocalteu’s phenol reagent for analysis-grade were obtained from Merck (Darmstadt, Germany). HPLC-grade acetonitrile, methanol, formic acid, ammonium formate were obtained from Merck (Darmstadt, Germany). (Sigma Aldrich, Saint Louis) was used as references for identification. Ultrapure water was produced by a Milli-Q apparatus (Millipore, Bedford, MA, USA).

Bacterial cells were grown at each salt concentration up to late log phase and centrifuged at 8960×g for 10 min at 4 °C. The pellet was washed with MilliQ water and then extracted overnight in 1 ml of 3% (aq) sulphosalicyclic acid. After removal of protein precipitates and debris by centrifugation, sample was prepared using Chinard’s reagent [33 (link)] and glacial acetic acid at boiling temperature for 1 h. Two milliliters of toluene was mixed in the sample plunged in ice and color was extracted for 15 min. Absorbance of colored compound was observed using toluene as blank at 520 nm. Standard curve was prepared using L-Proline (10–100 μg/ml) (Merck Millipore, Germany).

The proline content in wheat leaves was estimated according to Shah and Dubey [19 (link)]. Fresh leaf samples (0.2 g) were homogenized in 5 mL of 3% aqueous sulphosalicylic acid and then centrifuged at 12,000 rpm for 10 min. Acid ninhydrin (2 mL) and glacial acetic acid (2 mL) were added to 2 mL of supernatant. The mixture was boiled in a water bath at 100°C for 1 h and then extracted with 4 mL of toluene. The absorbance of chromophore was measured at 520 nm using toluene as a blank. L-Proline (Merck) was used to construct the standard curve. The proline concentration (μg/g FW) was calculated as follows: proline content (μg) × extraction solution volume (mL)/sample volume (mL) × fresh leaf sample weight (g).

Chondrogenic differentiation of hMSC-collagen microspheres was induced in the absence of serum using the well-established culture condition [11 (link)] by replacing culture medium with chondrogenic differentiation induction medium (CM) at the third day after encapsulation. CM was defined as Dulbecco’s modified Eagle’s medium-high glucose (DMEM-HG), supplemented with 10 ng/ml recombinant human TGF-β3 (Merck, Darmstadt, Germany), 100 nM dexamethasone (Sigma, St. Louis, MO, USA), 0.1 mM L-ascorbic acid 2-phosphate (Fluka, St. Louis, MO, USA), 6 mg/ml insulin (Merck), 6 mg/ml transferrin (Sigma), 1 mM sodium pyruvate (Gibco, Grand Island, NY, USA), 0.35 mM L-proline (Merck), 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine) and 1.25 mg/ml bovine serum albumin (BSA) (Sigma). CM was freshly prepared and regularly changed every 2 days for 4 weeks.

A pure standard solution of n‐alkanes (from n‐C7 to n‐C30) for linear retention indices (I^T) calibration and system quality control was from Merck (Milan, Italy) and prepared in toluene at the concentration of 100 mg L⁻¹.
The internal standard (IS) 1,4-dibromobenzene (from Merck, Milan, Italy) solution was prepared in toluene at a concentration of 10 g L⁻¹.
Pure reference standards used for identity confirmation of pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, 2-ketoglutaric acid, 3-hydroxybutyric acid, fumaric acid, 2-keto-3-metilvaleric acid, aspartic acid, hippuric acid, citric acid, uric acid, l-alanine, l-valine, l-leucine, l-proline, glycine, l-threonine, l-tyrosine, l-phenylalanine, l-isoleucine, l-methionine, l-cysteine, l-ornithine, l-tryptophan, xylitol, ribitol, fructose, galactose, glucose, mannitol, myo-inositol, glycerol, palmitic acid, stearic acid, and creatinine were from Merck (Milan, Italy).
Derivatizing agents O-methyl hydroxylamine hydrochloride (MOX), N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and LC-grade pyridine, n-hexane, dichloromethane, and toluene used as solvents were all from Merck (Milan, Italy).