Bicinchoninic acid solution

Proteins were isolated using the NucleoSpin^® RNA/Protein kit (MACHEREY-NAGEL). Protein contents were measured using Bicinchoninic acid Solution (Sigma, USA) and Copper (II) sulfate solution (Sigma, USA). Protein were separated on 12% or 8% SDS-PAGE gel. Western blotting of SDS-PAGE gels was performed using standard methodology. Primary antibodies were diluted at 1:1000 in 1% skim milk in PBST. Primary antibodies used in this study were LY6K (Santa Cruz Biotechnology, USA), ERα (Santa Cruz Biotechnology, USA), Argonate2 (abcam^®, UK) and β-actin (Bethyl Laboratories, US). β-actin was used as a loading control. Immunoreactive proteins were detected by horseradish peroxidase-conjugated secondary antibodies and the enhanced chemiluminescence reagent, EzWestLumi plus (ATTO, JAPAN).

Stock bovine serum albumin (BSA) standard (Sigma-Aldrich, Saint Louis, MO, USA) was prepared at a 2-mg/mL final concentration in water. Eight BSA samples with concentrations ranging from 0 to 2000 µg/mL were prepared by dilution for the standard curve. The bicinchoninic acid (BCA) working reagent was prepared by mixing 10 mL of solution A (Bicinchoninic Acid solution, Sigma-Aldrich) with 200 µL of solution B (4% w/v CuSO₄·5H₂O in water). The blank and protein samples were mixed with the resulting BCA working reagent at a 1:8 ratio; thus, 25 µL of each peptide or BSA or blank samples were mixed with 200 µL of BCA working reagent and were added to 96-microplate wells. The plate was incubated at 60 °C for 15 min and, after 5 min cooling to room temperature, the absorbance of all wells was measured at 562 nm using an ultrafast BioTek Synergy H1 plate reader (Winooski, VT, USA). All absorbance values were blank subtracted. Peptide concentration was calculated by interpolation using a standard curve (absorbance versus protein concentration) obtained with the eight BSA samples.

OCR was assessed using a Seahorse XF96 flux analyzer (Seahorse Bioscience). KG1 (75 × 10³ cells/well) and TEX (1 × 10⁵ cells/well) cells were cultured in a 96-well XF96 culture plate (Seahorse Bioscience) coated with BD Cell-Tak (BD Biosciences) in their regular growth medium for 24 h with or without R406. An hour prior to the analysis, cells were washed with PBS and resuspended in XF base medium (pH 7.4) (Agilent Technologies) supplemented with glucose (5 mM), L-glutamine (1.6 mM), and pyruvate (1 mM). Plates were incubated for 1 h at 37 °C in a CO₂-free incubator and transferred to the XF96 analyzer. OCR was measured at baseline and after the injection of oligomycin (1 µg/ml), FCCP (1.5 µM for TEX and 1.25 µM for KG1), rotenone/antimycin A (1 µM). After measurements, cells were washed with PBS, lysed in 10 μL of 0.1% Triton/PBS solution and frozen at −80 °C overnight. Thereafter, the protein concentration in each well was measured using BCA (Copper(II) sulfate solution and Bicinchoninic Acid solution, Sigma-Aldrich). Data were normalized to protein concentration and analyzed using Wave 2.0 software (Agilent Technologies).

DMEM, PBS (sterile), sodium pyruvate, penicillin/streptomycin, protein standard, bicinchoninic acid solution and copper (II) sulfate solution for total protein determination were purchased from Sigma-Aldrich (Taufkirchen, Germany); fetal bovine serum and glutamine were from Biochrom (Berlin, Germany). Resazurin was from Sigma Aldrich (Taufkirchen, Germany) and Alamar blue kit from BioRad (Feldkirchen, Germany) (BUF 012-B). LDH-Kit was purchased from Sigma-Aldrich (MAK066-1KT) with an additional standard from Cayman-Chemical (Michigan, USA). For the GSH enzymatic recycling method potassium dihydrogen orthophosphate, dipotassium hydrogen orthophosphate and Triton X-100 were aquired from Carl Roth (Karlsruhe, Germany). Sulfosalicylic acid was purchased from Sigma-Aldrich (Taufkirchen, Germany). DTNB, ß-NADPH, Glutathione reductase and GSH were purchased from Merck (Darmstadt, Germany). Diethiothreitol and Monobrombimane were from Sigma Aldrich (Taufkirchen, Germany).