The N-cyclin B-Luc counterscreen. (A) Titration results of reference control MG132 on HeLa cells transiently transfected with either the K328M-Wee1-Luc (●) or the N-cyclin B-Luc (○) expressing construct. MG132 was tested as a 10-point, 3:2 serial dilution starting at 30 μM. Data points show mean ± SD (n=3). (B) Scatter plot of the percent activation of the selected 1,090 primary hits measured in the Wee1 confirmation assay (X-axis) vs. the N-cyclin B-Luc counterscreen (Y-axis). Compounds were tested in triplicate at a nominal screening concentration of 5 μM. (C) Example of titration results of a false-positive, non-selective compound. Compound SID14743125 was titrated in a 10-point, 3:1 serial dilution starting at 50 μM in the Wee1 (○), Cyclin B (□) and viability (◇) assays. Error bars represent the standard deviation (n=3).