Victor x5

The total ATP content from synchronized human skin fibroblasts was determined using a bioluminescence assay (ViaLighTM HT; Cambrex Bio Science, Walkersville, MD USA) according to the instruction of the manufacturer. Cells were plated in 8 replicates into a 96-wells cell culture plate at a cell density sufficient to reach 40–50% of confluency on the following day. The enzyme luciferase, which catalyzes the formation of light from ATP and luciferin was used. The emitted light is linearly related to the ATP concentration and is measured using a multilabel plate reader VictorX5 (Perkin Elmer).
To measure NAD⁺ and NADH, the two molecules were separately extracted using an acid-base extraction method (HCL 0.1 mol/l–NAOH 0.1 mol/l). To determinate both NAD⁺ and NADH, an assay was used that is based on passing the electron from ethanol through reduced pyridine nucleotides to MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] in a PES (phenazine ethosulfate)-coupled reaction resulting in the formation of a purple precipitate (formazan) that, once dissolved, can be quantified at 595 nm (VictorX5, Perkin Elmer).

The cell viability assay was performed as previously described [9 (link)]. Briefly, the HepG2 and Huh7 were plated at 5 × 10⁴ cells/well (1.0 × 10⁴ cells/well for the PF cells) in a 96-well plate, followed by treatment with β-S, β-SG, or camptothecin at various concentrations of 40, 20, 10, 5, 2.5, 1.25 μg/mL after 24 h of incubation. After 48 h of further incubation, the treated cells were washed with PBS twice and a 100 μL fresh medium was added into each well. Afterward, a 100 μL of CellTiter-Glo^® Luminescent reagent (Promega, Madison, WI, USA) was added to each well to measure the cell viability with a luminescence signal using a microplate reader (Perkin Elmer, Victor X5, Victor X5, Norwalk, CT, USA). The cell viability percentage was calculated by comparing the absorbance values between treated and untreated cells. The experiments were repeated at least three times for the statistical analysis. To further evaluate the morphology of the cells, optical microscopy was also used during the experiment.

To measure dimer exchange by time-resolved Förster resonance energy transfer (tr-FRET), 2 aliquots of ER-LBD were site-specifically labeled (20 (link), 43 (link)), one at C417 with 30 equivalents of a thiol-reactive biotin derivative (Biotin-dPEG3-MAL, Quanta Biodesign), to which was added the donor fluorophore, a streptavidin-terbium chelate (SaTb, Invitrogen), and the other labeled at C530 with the acceptor fluorophore fluorescein (iodoacetamidofluorescein, Invitrogen). They were separately incubated with 1 × 10^–6 M of ligand for 0.5 hours. The two ER-ligand complexes were mixed together at concentrations of 2 nM ER-SaTb and 10 nM ER-fluorescein, and aliquots were taken with time and measured for the development of the FRET signal. Individual aliquots were measured at each time point to avoid bleaching artifacts. As the dimers exchange, there is a development of the FRET signal only when dimers have both donor (terbium complex) and acceptor (fluorescein) fluorophores (20 (link), 43 (link)). Measurements were performed, in 96-well black plates, with a PerkinElmer Victor X5 plate reader using an excitation filter at 340/10 nm and emission filters for terbium and fluorescein at 495/20 and 520/25 nm, respectively, with a 100-microsecond delay. Diffusion-enhanced FRET was determined by a parallel incubation without biotinylated ER-LBD and subtracted as a background signal.