Cpi 203

A2058, 1205Lu, C8161, SK-MEL 2, and SK-MEL 28 cells were pretreated with or without 50 μM sodium ascorbate for 72 hours. On a 384-well drug plate, a 3-fold serial dilution of BETi including BI-2536, CPI-203, BET-151 (Selleck chemicals, Houston, TX) or JQ1 (kindly provided by the Bradner lab, Dana-Farber Cancer Institute, Harvard University) were prepared with a starting concentration of 10 μM. On the day of treatment, BETi were simultaneously transferred to the cell plates with a 384-pipettor head using a FLIPR tetra instrument (molecular devices, Sunnyvale, CA). Cell viability was measured by CellTiter-GLo assay. Each concentration of BETi was plotted against percent cell survival. EC₅₀ values were calculated from 4-parameter fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value.

In vitro cell cytotoxicity assays were performed with CellTiter-Glo Luminescent Cell Viability Assay (Promega), according to the manufacturer’s instructions. Cells were seeded to opaque-walled black clear-bottom 96-well plates (Thermo Fisher Scientific) and incubated overnight. The next day, indicated concentrations the drugs being tested were added Carboplatin (Patterson Veterinary Supply, 07-890-7778), Cisplatin (Patterson Veterinary Supply, 07-893-4099), Prexasertib (Selleckchem, S71178), Olaparib (Selleckchem, S1060), Niraparib (Selleckchem, S7625), OTX015 (MK 8628/ Birabresib) (Selleckchem, S7360), CPI-203 (Selleckchem, S7304), (+)-JQ1, BET bromodomain inhibitor (Abcam, ab146612) for 72 hours. For synergy analysis of Prexasertib and Olaparib treatment (Figure 4A–B), cells were plated as above. The Bliss synergy score was calculated using SynergyFinder: a web application for analyzing drug combination dose-response matrix data (76 (link)). Luminescent Cell Viability Assay using CellTiter-Glo (Promega, G7571) was performed as per manufacturer’s instructions. IC50 values were determined using Graphpad Prism 8 (see Supplemental Table 1).