Celltiter blue

Assays assessing cytotoxicity of β-lapachone and derivatives were performed by CellTiter-Blue assay (Promega) according to the manufacturer’s instructions. Briefly, adherent cancer-cell lines were plated at 20,000 cells per well in 96-well plates or suspension cell lines were plated at a concentration of 2 × 10⁵ cells ml⁻¹ in 24-well plates. Compound stocks in media were made by dilution of DMSO stocks containing the compound of interest to obtain a final in-plate DMSO concentration of only 0.1%. Incubation was performed for 48 h. Following incubation, media was replaced with media containing CellTiter-Blue (Promega) in 1:10 dilution and the plates were incubated for 1.5–4 h. The fluorescence of the plates was recorded (excitation, 555 nm; emission, 585 nm; MiniMax i3x Reader, Molecular Devices). Cell viability was calculated by division of the fluorescence intensity of treated wells by that of the calculated average fluorescence intensity of replicate negative control wells containing cells with 0.1% DMSO only. IC₅₀ values were calculated using GraphPad Prism 8 software.

We validated the resazurin microtitre cell viability assay (REMA) as a screening method, REMA (CellTiter-Blue, Promega, Madison, WI, USA) was used to determine the antimicrobial activities of the drugs.¹¹ (link) Log phase M chimaera cultures were diluted to 1 × 10⁵–5 × 10⁵ CFU/mL, inoculated into a series of drug-free wells containing 2% v/v dimethyl sulfoxide (Sigma Aldrich, St Louis, MO, USA) and into wells containing 2·5 μg/mL rifampicin (50 times the minimum inhibitory concentration [MIC]; Sigma Aldrich, St Louis, MO, USA), and incubated at 37°C for 7 days. A 2% dimethyl sulfoxide concentration was selected to match the final dimethyl sulfoxide concentration of the Pathogen Box screen. To determine antimicrobial activity, CellTiter-Blue was added at a final concentration of 10% v/v and incubated for 16 h. Fluorescence was measured at excitation 580–640 nm and emission 520 nm, using a Glomax Discover plate reader (Promega, Madison, WI, USA). Fluorescence data were corrected for background using media-dimethyl sulfoxide bacteria-free controls. Experiments were repeated a minimum of three times and the datapoints were pooled.

The class I PI3K inhibitor, GDC-0941 (Axon Medchem), was used to treat bladder cancer cell lines and TERT-NHUC. The dose range chosen was based on previous studies that report IC₅₀ values of 0.28–0.95 μM for cell viability of solid tumor cell lines, as well as pharmacokinetic data available from phase I clinical studies that report a maximum of 2 μM GDC-0941 plasma concentration in patients [30 (link), 41 (link)]. Cell viability was assessed by CellTiter-Blue® (Promega) analysis of bladder cancer cell lines and TERT-NHUC subjected to GDC-concentrations from 0 to 2 μM. Cell viability was assessed by CellTiter-Blue® (Promega) analysis. 1000–4000 cells per well (number of cells determined to ensure that confluence was not achieved in untreated controls during the experiment) were plated in 96-well plates in five replicate wells and allowed to attach for 24 h before addition of 0–2 μM GDC-0941 in 0.1 % DMSO. After 72 h, 20 μl of CellTiter-Blue solution was added to the medium for 2 h and fluorescence read at 550 nm. Medium alone was used as a blank. Prism software (GraphPad Software, La Jolla, CA, USE) was used to calculate IC₅₀ values.
Cell cycle and apoptosis analysis of cells cultured with 1 μM GDC-0941 or DMSO only for 48 h was evaluated by flow cytometry as described [40 (link)]. All assays were done in triplicate and repeated at least three times.