Cytotox 96 non radioactive cytotoxicity

Cellular toxicity was measured by the lactate dehydrogenase (LDH) assay CytoTox 96^® Non-Radioactive Cytotoxicity (Promega, Spain, Madrid) following the manufacturer’s instructions. Briefly, 2.5 × 10⁵ PBMCs were seeded in 96-well plates and treated with the desired compounds for 48 h at concentration range of 5–50 µM. After the incubation period, PBMCs were lysed in 0.9% Triton X-100 (Promega, Spain, Madrid) for 45 min at 37 °C and 50 µL of LDH reagent (Promega, Spain, Madrid) was added for 30 min at room temperature, protected from light. The absorbance was read in a Berthold Plate Reader at 490 nm. All points were performed in triplicate. Absorbance values were interpreted as a measurement of cell viability. Concentrations resulted in cell viability above 80% [19 (link)]. Non-treated PBMCs were used as a viability control.

5 nm AuNPs
functionalized with tannate, citrate, and PVP, all purchased from
NanoComposix (San Diego, CA, USA), were used for this study. Phosphate-buffered
saline (PBS), F-12 nutrimix media, penicillin/streptomycin, 0.25%
trypsin-EDTA, fetal bovine serum (FBS), and RPMI 1640 were purchased
from Thermo Fisher Scientific (Paisley, UK). CytoTox 96 nonradioactive
cytotoxicity and GSH/GSSG-Glo luminescence assay kits were acquired
from Promega (Southampton, UK). Menadione, staurosporine, dimethyl
sulfoxide (DMSO), tris-base, glycine, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide), dichlorofluorescin diacetate (DCFDA), and sodium chloride
were purchased from Sigma-Aldrich (Dorset, UK). Bovine serum albumin
(BSA), acrylamide/bis-acrylamide, and ponceau were bought from Alfa
Aesar (Lancashire, UK). The Alexa fluor 488 Annexin V/PI cell death
reagent kit was purchased from Thermo Fisher Scientific (Paisley,
UK). The PVDF membrane and ECL were purchased from GE HealthCare (Buckinghamshire,
UK). USP7, USP8, USP10, UCHL-1, AKT, p-AKT, mTOR, GSK-3β, β-catenin,
p-β-catenin, GAPDH, caspase-3, caspase-9, and PARP primary antibodies
were purchased from Cell Signaling Technology (CST) (London, UK).

Cellular toxicity was measured by the lactate deshidrogenase (LDH) assay CytoTox 96® Non-Radioactive Cytotoxicity (Promega, Spain, Madrid) following manufacturer’s instructions. Briefly, 1 × 10⁵ MRC-5 cells were seeded in 96-well plates and treated with the desired compounds for 6 days in a concentration range (1–50 µM). After the incubation period, MRC-5 cells were lysed in 0.9 % Triton X-100 (Promega, Spain, Madrid) for 45 min at 37 ºC and 50 µl of LDH reagent (Promega, Spain, Madrid) was added for 30 min at room temperature, protected from light. Absorbance was read in a Berthold Plate Reader at 490 nm. All points were performed by triplicate. Non-treated MRC-5 cells were used as viability control.