Nad nadh glotm assay

NAD⁺ and NADH levels were determined using the NAD/NADH-Glo^TM Assay (G9071; Promega, Madison, WI, USA). On 6 cm plates, HEK293T (1.5 × 10⁶ cells), HFL-1 (3 × 10⁵ cells), and MRC-5 (3 × 10⁵ cells) were treated for 48 h with 25 µM EA or MDSA at 37 °C with 5% CO₂. HEK293T (2 × 10⁶ cells in 100 µL PBS), HFL-1 and MRC-5 (2 × 10⁵ cells in 100 µL PBS) were sonicated, and the supernatants were deproteinized using the 10 kDa spin column (Acrodisc® syringe filter, Pall Life Sciences). The supernatants (2 × 10⁴ cells in 100 µL PBS) were mixed with an equal volume of detection reagent in a 96-well plate, and the levels of NAD⁺ and NADH were determined using luminescent signals from the Biotek® multimode microplate reader (Agilent, Santa Clara, CA, USA).

NAD⁺/NADH-Glo^TM Assay (Promega, Madison, WI), NADP⁺/NADPH-Glo^TM Assay (Promega, Madison, WI), Reactive oxygen species (ROS)-Glo^TM (Promega, Madison, WI, USA), Glutathione (GSH)–Glo^TM Assay (Promega, Madison, WI, USA) were run based on same procedure. BT474, MCF7 ESR1^Y537S and T47D ESR1^Y537S cells were seeded at a concentration of 2 × 10³ cells/well in 96-well plates in corresponding phenol red-free media. Next day, cells were treated with endocrine agents alone or in combination with SEL in corresponding concentrations, and they were incubated with drugs for 24 h. All assays were run according to the supplier’s instructions for use of products. After 24 h incubation with drugs, luminescence values were measured using a Cytation5 plate reader (BioTek, Winooski, VT, USA). Standard curve and sample values were analyzed after subtraction of values from blank sample, and all statistical analyses were done by using Graphpad© Prism8 software (RRID:SCR_002798, GraphPad Software Inc., La Jolla, CA, www.graphpad.com). All experiment conditions had six technical repeats and experiments were repeated in six technical repeats at least for two times.

Cells grown in ammonium sulfate- or proline-containing minimal synthetic media for 16–18 h were harvested and washed with nanopure water. Spheroplasts were obtained from 10⁷ cells by resuspending cells in 1 ml zymolyase buffer (1.2 M sorbitol and 20 mM potassium phosphate, pH 7.4) containing 0.3 mg/ml zymolyase and incubating for 45 min at 30 °C. Spheroplasts were gently washed using zymolyase buffer and resuspended in 200 µl PBS buffer. NAD, NADH, NADP, and NADPH levels were determined using the NAD/NADH-Glo^TM assay and NADP/NADPH-Glo^TM assay kits (Promega; Cat. No. G9071 and G9081) according to the manufacturer’s protocol. Luminescence was recorded using a Synergy^TM Mx (BioTek) microplate reader. Values were normalized to buffer control.