Secondary assays confirmed the pro-survival effects of the ROCK inhibitor HA-1077 on OCT4 positive hESCs.

<p><b>A:</b> 320 candidate small molecules were rescreened in identical conditions to the primary assay. 2 small molecules circled in red, HA-1077 and T6140247, resulted in a significant (Z score ≥3 SD) improvement in viable Calcein positive objects. <b>B:</b> As a secondary assay, the 320 candidate molecules were screened at 1500 cells per well. After two days, cells were stained with the pluripotency marker OCT4 and counterstained with Hoechst 33342, in place of Calcein staining. The compounds identified in (<b>A</b>) are again circled. T6140247 is found clustered with other experimental samples (black) and negative control wells (red), while HA-1077 is found clustered with positive control wells treated with 10 µM HA-1077 (blue).