High-throughput screen for compounds affecting nuclear size. (A) Schematics of cell growth, treatment, and imaging methodology. (B) Schematics of outlier-based analysis methodology. Within each well, individual cells (left, bottom image) and their nuclei (left, top image) were masked. For nuclear and N/C ratio metrics, well-based distributions of individual cell metric values were computed for dimethyl sulfoxide (DMSO) (blue) and compound-treated wells (red, middle plot). The former was used to define outlier thresholds (blue arrows), from which the fractions of outlier cells (red-shaded regions) were computed for compound-treated wells (shown regions) and DMSO-treated cells (not shown). Blue and red asterisks show the mean value of the metric for DMSO- and compound-treated wells, respectively, defining the average-based analysis. For each cell line/time point, data were collected from all wells across the library/replicates (right plot, example shown is plate 14 for PC3, 6 h). Standard statistical analyses of each dataset were used to identify compounds (e.g., oxyphenbutazone) that increased the fractions of outlier cells with either small/large nucleus and/or N/C ratio beyond nonspecific effects of DMSO and inactive compounds (referred to as “hits”). From Tollis et al, ACS chem. biol. 2022
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