Sulforhodamine b dye

Cytotoxicity and proliferation were measured using the SRB assay. 250-3000 cells were plated per well in 96-well plates, treated with 1:4 or 1:2 dilution series of carboplatin (Sigma-Aldrich, Gillingham, UK), mitomycin C (Selleckchem, Houston, TX, USA) paclitaxel (Selleckchem), or olaparib (Selleckchem) and incubated for 5 days at 37°C. These were fixed using 25% trichloroacetic acid (Sigma-Aldrich), and stained with 0.4% w/v sulforhodamine B dye (Sigma-Aldrich). Dye was dissolved by addition of Tris pH 10.5, and optical density at 540nM was measured using a BP800 Microplate Reader (Biohit). Absolute IC₅₀ values were interpolated from concentration-response curves using Graphpad Prism v9.4.1.

Pterostilbene (>97% high-performance liquid chromatography (HPLC)-pure) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Pterostilbene was dissolved in dimethyl sulfoxide (DMSO) and further diluted in a sterile culture medium before use. DMSO, Sulforhodamine B dye, Dulbecco’s minimum essential medium (DMEM), trypsin/EDTA, phosphate-buffered saline (PBS), TRIS base, and acetic acid were also procured from Sigma-Aldrich (St. Louis, MO, USA). Alexa Fluor 488 and Alexa Fluor 647 donkey anti-rabbit IgG antibodies were purchased from Invitrogen (Grand Island, NY, USA). A TRIzol Plus RNA isolation and purification kit was obtained from Life Technologies (Invitrogen, Rockville, MD, USA), and real-time polymerase chain reaction (rt-PCR) primers, dNTP, reverse transcriptase, and Taq polymerase were obtained from Qiagen (Valencia, CA, USA).

Cells were seeded into a 96-well plate (Corning) at an optimal density to maintain final readouts in the linear range of the assay. After adhering overnight, the cells were treated zampanolide at the indicated concentrations in 0.5% DMSO (Fisher Scientific, Waltham, MA, USA) for 48 h. A separate time zero plate was fixed with 10% trichloroacetic acid (Sigma-Aldrich, St. Louis, MO, USA) to provide a readout of cellular density at the time of drug addition. After treatment, the cells were fixed with 10% trichloroacetic acid for a minimum of 1 h. The cells were then stained with sulforhodamine B dye (Sigma-Aldrich) for 30 min. Excess dye was washed off and the cellular-bound dye was solubilized in 200 µL of 10 mM Tris before reading the optical density at 560 nm on the Spectramax plate reader running SoftMax Pro 5.4 (Molecular Devices, San Jose, CA, USA). The percent growth or cytotoxicity of cells during the treatment period was determined as compared to the time zero plate (y = 0) and vehicle treated wells (y = 100) and replicates graphed with error bars representing SEM. Concentrations that caused a 50% inhibition of cell growth (GI₅₀) and total growth inhibition (TGI) were determined by non-linear regression analysis of the data using Prism (Graphpad, San Diego, CA, USA). Data are from 3 independent experiments each run in triplicate ± SEM.