Sodium metavanadate

B16-F10 cells at a starting density of 1 × 10⁶ cells were seeded into regular plates and treated as for the cell viability assay. Cells were harvested at intervals in a chilled lysis buffer containing 0.5 mM sodium-metavanadate, 1 mM EDTA and protease and phosphatase inhibitor cocktails (1:200), all purchased from Sigma–Aldrich Co. Cell lysates were boiled and subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis before being transferred to nitrocellulose membranes. The membranes were blocked in 5% low-fat milk for 1.5 hours at room temperature before being exposed to primary antibodies at 4°C overnight in a blocking solution. Appropriate horseradish peroxidase-conjugated secondary antibodies were used at a dilution of 1:5000 (anti-mouse and anti-rabbit IgGs; Sigma–Aldrich Co.) and visualized by enhanced chemiluminescence (Amersham Biosciences, Piscataway, New Jersey, USA). The films were scanned, and the pixel volumes of the bands were determined using NIH Image J software (Bethesda, Maryland, USA). For membrane stripping and reprobing, the membranes were washed in a stripping buffer (0.1 M glycine, 5 M MgCl₂, pH 2.8) for an hour at room temperature. After washing and blocking, the membranes were incubated with primary antibodies for nonphosphorylated or loading control proteins. All experiments were repeated three times.

Alexa Fluor647-ATP (cat. no. A22362) was obtained from Thermo Fisher Scientific. Trolox (cat. no. 238813), cyclooctatetraene (COT, cat. no. 138924), 4-Nitrobenzyl alcohol (NBA, cat. no. N12821), pyranose oxidase (POX, cat. no. P4234), bovine serum albumin (BSA, high purity, cat. no. A0281), dithiothreitol (DTT), catalase (cat. no. C100), creatine phosphate (PK, cat. no. P7936), adenosine triphosphate (ATP, cat. no. A2383), creatine phosphokinase (CPK, cat.no. C3755), MOPS, KCl, MgCl₂, K₂EGTA, HCl, KOH, Methanol, Glucose, DMSO, Sodium Orthovanadate, Sodium metavanadate, Methylcellulose, Adenosine diphosphate (ADP, cat no. A2754) were of analytical grade and purchased from Sigma-Aldrich (now Merck). The lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (cat. no 850355 C), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP, cat. no 890870 C) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (biotin-cap-DPPE, cat. no. 870277) were obtained from Avanti Polar Lipids. Para-aminoblebbistatin (PAB) was obtained from Motorpharma (Hungary). Any other biochemical reagents were of analytical grade and purchased from Sigma-Aldrich (now Merck).

Sodium metavanadate (99.9% NaVO₃) was purchased from Sigma-Aldrich, hydrochloric acid (36.5–38%, HCl) and citric acid (99% C₆H₈O₇) were purchased from Fischer. Deuterium oxide (D₂O, 99.9%) was purchased from Cambridge Isotope Laboratories, Inc. All reagents and other chemicals was purchased from Sigma-Aldrich. Vanadium complexes were prepared according to literature (Samart et al., 2014 (link); Sanchez-Lombardo et al., 2016 (link)).

Cells were cultured in a 225 cm² flask (Falcon, 353138) to a maximum confluency of 95%, then cells were rinsed twice in PBS and lysed in 12.5 ml of lysis buffer per flask (50 mM Tris pH 7.8, 1% NP40 (Sigma-Aldrich, 74385), 120 mM NaCl (Sigma-Aldrich, 71380), 25 mM NaF (Merck, 1.06450.0025), 40 mM β-glycerophosphate disodium salt (Sigma-Aldrich, 50020), 100 µM sodium metavanadate (Sigma-Aldrich, 590088), 1 mM DL-DTT (Sigma-Aldrich, 43815), 100 µM phenylmethyl sulfonyl fluoride (Sigma-Aldrich, P-7626), 1 mM benzamidine (Sigma-Aldrich, B-6506), 1 µM microcystin (Alexis Biochemicals 350-012-M001)). The lysates were centrifuged at 4 °C for 10 min and total protein was quantified and adjusted to 0.5 µg µl⁻¹. Then, 100 µl of cell lysates was then plated in 96-well plates, the lysates were treated with HRO761 starting at 10 µM using the HP Dispenser (software D300eControl) and then incubated for 72 h at 20 °C. After incubation, the plates were used for in WRN enzyme-linked immunosorbent assay (ELISA) analysis as described below. PS₅₀ values are the levels of HRO761 needed to stabilize the WRN protein 50% over DMSO control.

rSmPGM activity was measured after 10 min preincubation in the absence or presence of either 1 μM or 10 μM sodium metavanadate (Sigma-Aldrich). PGM activity was then initiated by adding 3PG (5 mM) and enolase (0.2 μg/reaction), and the reaction was monitored as described above, in triplicate.

All chemicals were analytical reagents and used without further purification. Bismuth nitrate pentahydrate ((Bi(NO₃)₃∙5H₂O, ≥98%) and sodium metavanadate (NaVO₃, ≥98%) from Sigma-Aldrich (St. Louis, MO, USA), nitric-acid (HNO₃, 69%) from Merck, oxalic acid (H₂C₂O₄·2H₂O, ≥99.9) from Scharlau, rhodamine B (C₂₈H₃₂ClN₂O₃) from ReAnal (purity ≥99.9%), cooper chloride dihydrate (CuCl₂·2H₂O, 99%) from Alfa Aesar (Karlsruhe, Germany)and high purity deionized water were used throughout the whole work.