Figure 4. Analysis of fitness multiplexed cell lines and identification of cell line-specific fitness phenotypes.

A–B. Hit confirmation by combinatorial analysis of multiple fitness indicators. Recorded fluorescence and luminescence intensities were normalized to the plate median and plate replicates were averaged for further analysis. Cell fitness phenotypes were selected from ranked mean values with small values indicating strong decrease in fitness. 25, 50 and 75 top-ranking fitness phenotypes were selected for both cell lines as well as for randomly generated values from all fitness indicators and the intersection (in %) of phenotypes identified in two (A.) and three (B.) channels was calculated as measure of hit confirmation. The fraction of intersecting phenotypes from two or three channels is comparable for both cell lines and differs significantly from randomly generated numbers. These results indicate that the fitness indicators are strongly linked and that combination of multiple fitness readouts increases confidence for hit selection. C–D. Venn-diagrams for the three fitness indicators constructed from selected top 25 fitness phenotypes of both cell lines. E. Identification of cell-line specific fitness phenotypes. To identify genes relevant to HD from the proof-of-principle study we identified siRNAs which showed strong cell fitness effects in the Huntingtin mutant cell line (HTT-Q79) but not in wild type cells (HTT-Q23). A total of 6 siRNAs were identified (see grey area in overlapping circles).