Optimal HTS assay conditions were established based on previous work published elsewhere[15 (link), 22 (link)–24 (link)] and adapted to our system conditions described in Online Resource 1. Cells were exposed to each of the 2,700 medicinally active and structurally diverse compounds contained in the Selleck Bioactive and Selleck FDA-approved libraries (SelleckChem, Houston, TX). Cells were automatically counted, seeded in 384-well plates, and cultured for 24 h. Next, compounds were diluted in 0.5% DMSO (v/v) and transferred into the well plates using an Agilent Bravo Liquid Handler, resulting in a final concentration of 4.8 μM. All plates also included positive (melphalan) and negative (vehicle) controls; melphalan was used based on its potent and reproducible in vitro cytotoxicity[21 (link), 25 (link)]. After 72 h of drug exposure, cell viability was determined[15 (link), 24 (link)]. Compounds that showed > 70% cytotoxic activity at 4.8 μM were considered pharmacologically active against retinoblastoma.