Identification of potential chemical compounds enhancing generation of enucleated cells from immortalized human erythroid cell lines

摘要

a Schematic of experimental procedure. Ten thousand HiDEP-1 cells (HiDEP) per well were cultured with 0.5 μM or 10 μM of chemical compounds dissolved in DMSO for 4 days. Cells were then stained with SYTO 16 and SYTOX Red, and subsequently subjected to Cellomics Arrayscan analysis and cytospin analysis. b Theoretically expected output patterns of cell statuses based on the three fluorescent signals. KuO: Kusabira Orange, representing an expression of HPV-E6/E7; SYTO 16: cell permeant dye that stains nuclei of both live and dead cells; SYTOX Red: cell non-permeant dye that stains nuclei of dead cells. c Representative fluorescence images of HiDEP stained with two DNA dyes. From left to right: bright-field, Kusabira Orange, SYTO 16, SYTOX Red, and Merged image. The white arrow shows an enucleated cell that is positive for KuO but negative for SYTO 16 and SYTOX Red. Scale bars represent 20 μm. d List of chemical compound libraries used in this study. CBT library: in-house novel and nondisclosed chemicals from Chemical Biology and Therapeutics group, Lund University. e Result of the compound screening. All chemical compounds were tested at 0.5 μM and 10 μM. The X-axis represents relative numbers of KuO+ cells and the Y-axis represents a relative number of SYTO 16− SYTOX Red− cells. The values are calculated as fold change compared to the mean value of DMSO controls in the same test plate. Light yellow area marks chemical compounds that achieved more than 9.0-fold increase in the enucleation frequency and not <0.25-fold in the total cell number therefore selected as hits. Black dots, tested chemical compounds; blue dots, DMSO controls; orange dots, HDACi. f Representative images of cells captured on Cellomics Arrayscan. Bright-field (upper panel) and combinatory staining (Merged, lower panel) of cells treated with DMSO, a representative non-hit compound and one of hit compounds are shown. The white arrows show enucleated cells. Scale bars represent 20 μm.