Development of an HTS system to discover compounds that facilitate MK maturation. (A) Schematic illustration of the screening procedure. From an initial pool of 5245 compounds, 67 compounds were selected based on the Venus fluorescence intensity in the first screening and were subjected to a second screening for further validation. Twenty-eight compounds improved MK maturation and subsequent PLP production. (B) Strategy for screening compounds that promote MK maturation based on the visualization of β1-tubulin–Venus expression. β1-tubulin–Venus imMKCLs (30 000 cells per well) were cultured in 96-well plates for 7 days in the presence of drugs. Venus fluorescence intensities were detected using an ArrayScan. (C) Representative fluorescent images of β1-tubulin–Venus imMKCL maturation in the absence or presence of SR1. The green fluorescent cells represent Venus-expressing cells. (D) Quantified Venus intensity of β1-tubulin–Venus imMKCLs cultured in the absence or presence of SR1. β1-tubulin–Venus imMKCLs were cultured in 96-well plates for 7 days in the absence or presence of SR1 (0.75 μM). (E) Formula for calculating relative values from measured ones. Data were normalized to the plate negative (Vehicle) and positive (SR1) controls. (F) Relative PLP production from imMKCLs treated with candidate compounds. β1-tubulin–Venus imMKCLs (2 × 10⁵) were incubated for 7 days in the absence or presence of candidate drugs at their optimal concentrations (0.4-10 μM), after which CD41⁺CD42b⁺ PLPs were detected and counted using flow cytometry. Values were normalized to PLP production in the presence of SR1. Data are expressed as the mean ± SD from 3 to 5 independent experiments. ***P < .001 vs Vehicle, Student t test.