Primary screen of 44,000 compounds and confirmation of activity. (A) Example screening results for the first approximately 10 K compounds. Viability of cells exposed to 500 ng/mL dox and 5 μM compound is indicated on the y-axis, normalized to control wells, not treated with doxycycline. Red circles indicate viability scores ≥3 SD above the mean viability. (B) Retesting of compounds at 1.67 μM, 5 μM, and 15 μM. (C) Schematic of workflow. Primary HTS identified rare ‘hits’, which were then collected in very small volumes into cherry-picked plates. These were then used for secondary screens. Using data from the secondary screens, individual compounds are identified for deeper study.