Figure 1.
High-Throughput Screening
(A) Schematic of high-throughput screening for generating contractile smooth muscle cells and restenosis drug discovery. The MYH11-NLuc-Tom reporter cell line was differentiated into mesoderm by E8BAC medium (E8 medium [Chen et al., 2011] supplemented with 5 ng/mL BMP4, 25 ng/mL activin A, and 1 μM CHIR99021) for 2 days and then treated with 50 ng/mL FGF2 and 20 ng/mL BMP4 in E6 medium (E8 medium minus FGF2 and TGF-β1) for another 2 days. The cells were passaged at day 4 and seeded on the 96-well plate for screening (2 × 106 cells/plate). The small molecules were added to the medium from day 4 to day 14. d, day; ES, embryonic stem; SMCs, smooth muscle cells; Tom, tdTomato.
(B) Screening results. Two screens were performed with a total of 4,804 small molecules. The luciferase assay results of individual small molecules were normalized to the average reads of all samples for each batch. The small molecules were selected for further analysis when the normalized reads were greater than “average + 3× SD.”
(C) Optimizing the concentration of the selected small molecules.