384 well white culture plates

Cell viability was assayed in 2D-adherent or ultra-low adherent 3D-spheroids using CellTiter-Glo (Promega)^{6 (link),15 (link)}. Luminescence was measured on an Envision 2104 plate reader (PerkinElmer) and Fluostar Omega Reader (BMG Labtech) and data was analyzed using GraphPad PRISM v8. Synergy assessments were performed using CellTiter-Glo (Promega) with the following modifications to the protocol described in the ref. ^{34 (link)}. In brief, EWS502 cells were plated at 1000 cells per well in 50 µL of appropriate media in 384-well white culture plates (Corning) allowed to adhere overnight, and 100 nL of compounds were added using a Janus Workstation pin tool (PerkinElmer) for 72 h. Cell viability was measured by addition of 10 µL of CellTiter-Glo (Promega), followed by incubation for 15 minutes at room temperature. Luminescence was measured on an Envision 2104 plate reader (PerkinElmer) and data was analyzed using GraphPad PRISM v8.

Dual luciferase assays were performed using 293FT FKBP12^WT-Nluc and FKBP12^F36V-Nluc cells^{6 (link)}. In brief, cells were plated at 2000 cells per well in 20 µL of appropriate media in 384-well white culture plates (Corning), allowed to adhere overnight, and 100 nL of compounds were added using a Janus Workstation pin tool (PerkinElmer) for 24 h at 37 °C. To evaluate Fluc signal, plates were brought to room temperature, 20 µL of Dual-Glo Reagent (Promega) was added for 10 min and luminescence was measured on an Envision 2104 plate reader (PerkinElmer). Subsequently, 20 µL of Dual-Glo Stop & Glo Reagent (Promega) was added for 10 min and luminescence was again measured to capture Nluc signal. DMSO-normalized ratios of Nluc/Fluc signal was analyzed and plotted using GraphPad PRISM v8^{6 (link)}.