Robustness and sensitivity of the experimental system at the level of primary screening. Stable HeLa cells were cultivated on 384-well siRNA arrays for 36 h, followed by the induction of expression of SH4-HASPB-GFP and SH4-YES1-mCherry. Nuclei were labeled and cells were subjected to live cell fluorescence microscopy in areas of immobilized siRNAs. Enlarged image details from primary screening data after transfection of a scrambled siRNA (A,B) and an siRNA targeted against the coatomer subunit COPB1 (C,D) are shown. Scores of all individual control experiments from primary screening were calculated using the image analysis tool described and are shown for SH4-HASPB-GFP (E,G) and SH4-YES1-mCherry (F,H), respectively. Negative controls (scrambled siRNA) are shown in panels E and F. Positive controls (siRNA targeted against COPB1) are shown in panels G and H. Scores below the defined hit threshold lie between the dashed lines within the gray shaded area.
