Figure 5

Phenotypic analysis and small molecule compound follow-up. (A) Time of death (days) for each well that died within the time frame of the screen, averaged over the three screens. 0 represents that the cells died within the 20 minutes between small molecule addition and initial imaging. 1 represents that cells died before 24 hours post-addition (i.e., between 12 hours and 24 hours). If cells did not die, they are not represented in this graph. Nuclei count was unreliable for cells which died, as there was debris created which fluoresced. Thus, time of death was manually quantified. (B) Phenotypic analysis of cardiomyocytes over three screens given 429 small molecules. (C) Example of a cluster of approximately 7–8 healthy cardiomyocytes. Small molecule: PD318088. (D) Example of a cluster of cardiomyocytes that are especially unhealthy, giving the cells a “spindly” appearance. Small molecule: AT7867. (E) Example of an elongated cardiomyocyte (arrow). Note cardiomyocytes directly above the elongated cardiomyocyte that have normal appearances. Small molecule: WAY-600. (F) Example of a particularly large cardiomyocyte (arrow). Small molecule: PD318088. (G) Example of a cardiomyocyte with stress-fiber like striations on its edge (arrow). Small molecule: APTSTAT3-9R. (H) Three small molecules identified by screen as potential regulators of hiCM proliferation. (I) Three small molecules from (H) tested at 0.01, 0.1, 1, and 10 µM (i.e., 10, 100, 1000, and 10000 nM) over five independent experiments. Shown as mean. (J) Binucleation proportion does not change when cells are treated with these small molecules at their most effective concentrations (Palomid-529 5 µM, SB216763 3 µM, and Acadesine 3 µM), measured post-fixation stained with β-catenin. N = 6624 total cells over three independent experiments; mean +/− SEM. NS by One-Way ANOVA.