Figure 2. Either Firefly or Renilla luciferase can function as p53-dependent reporters.

(A) The ability of Firefly and Renilla cDNAs to serve as reporters for p53 transactivation was examined by placing them downstream from the moderate p53 RE derived from the PUMA promoter in isogenic strains. The values indicate the fold induction measured over an empty vector. Presented are average and standard deviations of three replicates relative to optical density of the cultures measured at different times (T in hrs) after switching cultures to galactose-containing medium. (B) Dual luciferase reporter assay with a strain expressing WT p53 and containing the Firefly luciferase as p53 reporter gene and the Renilla luciferase as constitutive reporter. Presented are the average and standard error of the Firefly luciferase activities normalized for Renilla and compared to empty vector at various time points after shifting 100 µl yeast cultures to galactose-containing media in the 96-well plate format. (C) Comparison of relative induction using measurement of protein from 2 ml cultures vs direct permeabilization of cells in a 384 well format following transfer from a 96-well growth plate, as described in the text and the Materials and Methods section. Relative transactivation capacities of WT p53 and the R282Q mutant in the “2 ml vial”experimental set-ups were measured using either protein extraction or permeabilization. Experiments were conducted using 0.032% galactose inducer, unless specified otherwise. Error bars plot the standard error of four biological replicates.