Cs0170

The total thioredoxin reductase (TrxR) activity in cells was measured by using a commercial colorimetric assay kit (Sigma Aldrich CS0170) based on the reduction of 5,5’-dithiobis (2-nitrobenzoic) acid (DTNB) with NADPH to 5-thio-2 nitrobenzoic acid (TNB) at 412 nm. Since several enzymes present in biological sample can reduce DTNB, the kit also contains an inhibitor solution of mammalian thioredoxin reductase. This inhibitor allows determining the reduction of DTNB due only to TrxR activity. A2780 cells were treated for 24 h with concentrations corresponding to their 72 h-exposure IC₅₀-doses and then lysed with RIPA buffer (50 mM Tris–HCl pH 7.0, 1% (v/v) NP-40, 150 mM NaCl, 2 mM ethylene glycol bis(2-aminoethylether)tetra-acetic acid, 100 mM NaF) supplemented with a cocktail of protease inhibitors. The protein concentrations in the cell lysates were determined by Bradford protein assay kit (Bio-Rad Laboratories) according to the manufacturer’s instructions and then 30 µg of proteins were used for the assay. Results were normalized to the cellular protein content. The experiments were performed in triplicate (three independent biological replicates). The statistical analysis was carried out using one-way ANOVA test followed by Tukey’s multiple comparisons test using Graphpad Prism software v 6.0. P-value p < 0.05 was considered statistically significant.

Exponentially growing cells were seeded in 20 mm² Petri dishes at a density of 6 × 10⁵ cells/dish for 24 h. Thereafter, A2780 cells were treated with gold compound concentrations corresponding to their 72-h exposure IC₅₀ doses for 24 h. At this time, cells are viable as demonstrated by MTT time course experiments (Figure S9). At treatment, cells were lysed with RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 100 mM NaF, 2 mM EGTA, 1% Triton X-100) containing 10 μL/mL protease and phosphatase inhibitors (Sigma Aldrich). Lysates were centrifugated at 4 °C, 14,000 RPM for 15 min, and supernatants were collected. After protein quantification with Bradford Assay, 30 μg of proteins were used for the assay. TrxR activity was measured by using a commercial colourimetric assay kit (Sigma Aldrich CS0170) based on the reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) with NADPH to 5-thio-2-nitrobenzoic acid (TNB) at 412 nm. This kit also contains a solution of mammalian TrxR inhibitor. Experiments were performed in triplicate. Results were normalized to the cellular protein content and reported as the percentage of enzyme activity compared to the untreated cells (control).

A2780, IGROV1 and SKOV3 cells were treated for 24 h with Au(NHC) or Au(NHC)₂ concentration corresponding to their 72 h-exposure IC₅₀-dose. Cells were lysed in RIPA buffer containing a human protease inhibitor cocktail (Sigma-Aldrich), and 30 μg of proteins were used for the enzymatic assay. The TrxR inhibition was assessed using a commercial colorimetric assay kit (Sigma-Aldrich, CS0170) based on the reduction of 5,5’-dithiobis(2-nitrobenzoic) acid (DTNB) with NADPH to 5-thio-2-nitrobenzoic acid (TNB), according to the manufacturer's instructions. This kit also contains an inhibitor solution of mammalian thioredoxin reductase. Since several enzymes present in biological sample can reduce DTNB, the specific inhibitor is used to determine the reduction of DTNB due only to TrxR activity. Experiments were performed in triplicate. Results were normalized to the cellular protein content.