Fig. 4.

Synthetic lethal screen of 70 compounds. We used siRNAs in either A549 or HeLa cells to knock down one of seven target genes and then seeded each set of knocked-down cells on identical microarrays loaded with 70 different compounds. (a) To confirm that each siRNA was effective, we performed Western blots on small aliquots of cells that were simultaneously seeded on the microarrays. The right lane of the blots was loaded with lysate from cells transfected with siRNA for a target gene, and the left lane was loaded with lysate from cells treated with the same reagents without the siRNA. All gels were loaded with samples normalized for total protein. (b)We found four compound-siRNA pairs in which there was a significant difference in activity in the microarray format. Student's t tests were performed on cell density measurements taken at each distance from the center of the spots, and significant changes in cell density are highlighted in yellow. (c) We confirmed the macbecin II/TSC2 synthetic lethal effect by using a calcein acetoxymethyl ester viability assay in microtiter plates.