Mercuric chloride hgcl2

NaNO₃ was obtained from VWR international (Lutterworth, Leicestershire, UK). Sodium nitrite (NaNO₂), N-ethylmaleimide (NEM), Nonidet™ P 40 Substitute, mercuric chloride (HgCl₂) and 1 M hydrochloric acid (HCl) were obtained from Sigma-Aldrich Ltd. (Gillingham, UK). S-nitrosoglutathione (GSNO) was obtained from Enzo Life Sciences (Exeter, UK). Sodium iodide (NaI), potassium iodide (KI), iodine (I₂), acetic acid (99.5%), ethylenediaminetetraacetic acid (EDTA), vanadium (III) chloride (VCl₃), potassium ferricyanide, sulfanilamide and methanol were obtained from Fisher Scientific (Loughborough, Leicestershire, UK). Lithium-heparin tubes were obtained from Becton Dickinson UK Ltd. (Wokingham, UK). Cannulas were obtained from Becton Dickinson Insyte-WTM (Madrid, Spain). NO₃⁻-rich beetroot juice (BR; beetroot juice containing 6.4 mmol of NO₃⁻ per 70 ml; “Beet It Sport”) and NO₃⁻-depleted beetroot juice (PL; beetroot juice containing 0.04 mmol of NO₃⁻ per 70 ml) were obtained as gifts from James White Drinks Ltd. (Ipswich, UK). The reagents and buffers were made up in ultrapure water (18.2 MΩ/cm) unless otherwise indicated.

Shoot apices (1–1.2 cm) were collected from the Medicinal Plant Garden, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, India. Surface disinfection of the shoot apex explants was done following a series of treatments using autoclaved (at 1.1 kg/cm² pressure and 121 °C for 15 min) solutions of 0.03% (w/v) bavistin, 10% (v/v) Tween-20, 20% (v/v) sodium hypochlorite (NaOCl), 1% (w/v) cetrimide, ethyl alcohol (C₂H₅OH), and 0.1% (w/v) mercuric chloride (HgCl₂) (all these were obtained from Merck Life Sciences. Pvt. Ltd., India), carried out in the Laminar Air Flow cabinet (LAF). All of the sterilants were used for 5 min with the exception of ethyl alcohol, which was used for 30 s. After disinfection, the explants were taken out and the exposed basal portions of the shoot apices were trimmed and removed.

SH-SY5Y cells (American Tissue Culture Collection) were cultured in a humidified atmosphere with 5% CO₂ at 37 °C. D-MEM/F-12 (1:1) medium, supplemented with 15% FCS and 1% penicillin/streptomycin, was used as growth/proliferation medium. To induce neuronal differentiation, SH-SY5Y cells were cultured in serum-free Neurobasal™ medium with neural cell supplement (B-27), 10 µM retinoic acid, 1% L-glutamine and 1% penicillin/streptomycin (Gibco, Life Technologies) for seven days. All experiments were performed with differentiated, post-mitotic SH-SY5Y cells. HEp-2 cells (American Tissue Culture Collection) were cultured under the same atmospheric conditions in RPMI1640 medium supplemented with 10% fetal calf serum (FCS) and 5% supplement complete (SC). Cells were treated for four hours or as indicated with 25 µM (SH-SY5Y) or 60 µM (HEp-2) I-Hg, e.g., mercuric chloride (HgCl₂, Merck KGaA). These treatment protocols were established according to results from atomic absorption spectroscopy of nuclear cell fractions (Table 1) and titration of I-Hg concentrations that do not induce cell death (Fig. S1).