Fig. 1.

RNAi analysis of the E2F pathway in Drosophila cell culture. (A) Schematics of the reporter constructs. The E2F-dependent reporter (E2F-Luc) carries four copies of an artificial E2F site (4xE2F), a minimal HSP70 promoter (HSP), and the firefly luciferase gene (Luc). The Renilla control reporter contains the same HSP and the Renilla luciferase gene (RLu). The two constructs share the same plasmid backbone. (B) Effects of RNAi depletion of known E2F pathway components on the E2F-reponsive reporter. Drosophila S2* cells were transiently transfected with E2F-Luc and the control reporter and incubated with dsRNAs in 24-well plates. RNAi of dE2F1/dDP and Rbf1 considerably decreased and increased the E2F-dependent reporter, respectively. RNAi of Rbf2 or dE2F2 had little effect. The reporter responses remain constant during incubation days 4–8. Similar results were also obtained by using several other Drosophila cell lines (e.g., S2, Kc167, Dl2, Cl8).