Extended Data Fig. 1: Discovery of small molecule regulators of keratinocyte stem cell fate.

(a,b) Screening concept using calcium driven differentiation combined with TR-FRET assay for differentiation marker involucrin. The TR-FRET assay was optimized in pilot experiments in 384-well format and then miniaturized for the primary screen to 1536-well format with a Z’ mean 0.66 ± 0.18 over plates. (c) Screening flow chart starting with 108,000 compounds followed by several triaging selections leading to a final 110 active compounds. (d) HTS screen showing involucrin expression in the presence of all compounds tested at 1.25 µM (threshold for further validation was >30% reduction in involucrin expression). (e) Inhibition of involucrin (TR-FRET) expression versus cell number for 625 compounds in dose response. Compounds with >30% reduction in involucrin expression and <45% reduction in cell number at 10 µM were selected for further follow-up. Compound MoA annotation for most potent hit classes identified are shown: BET bromodomain inhibitor (red), Gamma secretase inhibitor (blue) or HDAC inhibitor (yellow). (f) 158 selected compounds (IC50 < 10 µM and <60% reduction in cell number at 10 µM in neonatal keratinocytes) validated in dose response on adult keratinocytes, correlating IC50 values (inhibition of involucrin expression) on adult versus neonatal cells. The middle gray line is the line of equality, where both IC50 values are identical, and the gray-dashed lines are one log unit above and below the line of equality (quisinostat; JQ1; I-BET151; I-BET762; DAPT). Color coding as in (e) with representative compounds per hit class labeled.