Fig. 2. Dup mRNA levels and E2F1/DP-dependent transcription are not increased during replication stress. (A,B) In-situ hybridization to Dup mRNA in a third-instar brain from wild-type (A) and Mcm6³ mutant (B) larvae. (C-F) E2F1/DP transcription-factor activity is not increased in brain cells with elevated Dup levels. Dup labeling (red) and anti-Myc labeling (green) to detect expression of the E2F1/DP-sensitive reporter ORC1p:ftz-GFP-Myc in control (C,D) or HU-treated (E,F) brain cells. (E) In HU-treated cells, Dup was often abundant in the nucleus and cytoplasm but was excluded from the nucleolus, which appears as an absence of staining in a spherical area in the nucleus. HU-treated cells did not have significantly elevated E2F/DP reporter activity (F) despite having high levels of Dup in the same cells (E). Images in A,B are bright field and those in C-F are composites of confocal sections. Scale bar, 50 µm (A,B), 10 µm (C-F).