Nah2po4

DMEM (high glucose, [4.5 g/L]), NaCl, FBS, and a BCA protein assay kit (Pierce, Rockford, IL, USA) were purchased from Euroclone (Milan, Italy); Na₃PO₄, MgSO₄, NaH₂PO₄, KH₂PO₄, and KCl were purchased from Carlo Erba (Milan, Italy); ferric citrate, Trypsin-EDTA-Solution (T4174) and Collagenasi type IA (C9891) were purchased from Sigma (St. Louis, MO, USA); the primary antibody for Axl (sc-1097) and GAS6 (N-20 sc-1936) were purchased from Santa Cruz (Heidelberg, Germany); the primary antibody for LC3-II was purchased from Cell Signalling (Danvers, MA, USA), and the anti-rabbit secondary antibody was purchased from GeneTex (Irvine, CA, USA); the anti-goat secondary antibody (ab6741) was purchased from Abcam (Cambrige, UK); Hepes Buffer Solution, PVDF membrane Invitrogen/Applied Biosystem (Milan, Italy); PE Annexin V Apoptosis Detection Kit I (559763) was purchased from Bioscience. An ApopTag Red In Situ Apoptosis Detection kit S7165 was purchased from Chemicon. All other reagents were obtained from Sigma (St. Louis, MO, USA).

All the chromatographic solvents were HPLC grade or LC-MS grade for the MS experiments. Acetonitrile, acetone, methanol and formic acid were purchased from Sigma Aldrich (Milan, Italy). Isotopically labelled compounds, rosmarinic acid-d7 and cinnamic acid-d5, were purchased from C/D/N Isotopes Inc. (Quebec, Canada). Pure anthocyanins were obtained from Polyphenol Laboratories AS (Sandnes, Norway) and Heparin from Schwarz Pharma (Milan, Italy). All chemicals were used without further purification. Ultra pure Milli-Q water (Merck Millipore, Billerica, MA, USA) was used for the preparation of all solutions. Phosphate buffered saline (PBS) was prepared as following: 6.03 mM Na₂HPO₄, 3.91 mM NaH₂PO₄ and 139 mM NaCl (Carlo Erba, Milan, Italy) were dissolved in MilliQ water (Millipore) and pH was adjusted to 7.4 with HCl.

Far-UV CD spectra were recorded on a Jasco J-710 spectropolarimeter. Samples were prepared using a Yfh1 concentration of 10 µM in 10 mM HEPES buffer at pH 7.5 and with a variety of salts: NaCl (Sigma-Adrich), KCl (Aldrich), MgCl₂ (Aldrich), CaCl₂ (J.T.Baker), NaF (May&Baker), NaH₂PO₄ (Carlo Erba), Na₂SO₄ (Sigma-Aldrich) and NaI (Carlo Erba). Baseline correction was performed by subtraction of the appropriate buffer spectrum. Thermal unfolding curves were obtained by monitoring the ellipticity at 220 nm using a Jasco J-815 CD spectropolarimeter equipped with a Jasco CDF-4265/15 Peltier unit. Measurements were repeated at least twice on independent protein preparations to ensure reproducibility of the results. All samples used Yfh1 at 10 µM in a 10 mM HEPES buffer at pH 7.5 and varying concentrations of NaCl, KCl (Fisher Scientific), MgCl₂, CaCl₂, FeSO₄, NaF (Sigma-Aldrich), NaH₂PO₄ (Acros Organics) and Na₂SO₄ (Merck). 2 mm path length cuvettes (Hellma) were used with a heating rate of 2°C/min in the temperature range 0–80°C. In the case of the ferrous salt rigorous anaerobic conditions were achieved by preparing the samples in a glove box, sealing the cuvette and performing the measurements under N₂ (g) flow.