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. 2010 Jun 14;189(6):981–996. doi: 10.1083/jcb.200910006

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© 2010 Buttitta et al.

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Figure 1. — Degradation of E2F1 activator complexes and a switch to E2F2 repressor complexes contributes to the CycE–Cdk2-resistant repression of E2F activity after exit. Clones overexpressing CycE–Cdk2 and negatively marked by CD2 (A and B) were generated using hs-FLP act>Gal4/UAS in e2f2^76Q1/G5.1 mutant wings. E2F transcriptional activity was monitored at the indicated stages using the PCNA-GFP reporter (A′ and B′). CycE–Cdk2 activity is temporarily able to activate the E2F reporter in the absence of e2f2 (A) but is unable to sustain E2F activity after 40 h APF (B). (C–E) GFP-marked e2f2-null mutant clones expressing CycE–Cdk2 were generated using the MARCM system and examined for ectopic mitoses by staining for PH3. Neurons are marked by expression of Elav. Ectopic mitoses in e2f2^−/− cells expressing CycE–Cdk2 are evident in Elav-negative cells in eyes from 24 to 40 h APF (36 h APF shown; C) but are no longer observed by 44 h APF (D). No ectopic mitoses are observed in e2f2^−/− cells expressing CycE–Cdk2 in wings after 36 h APF (E). (F–H) Clones expressing either GFP-E2F1^WT or GFP-E2F1^PIP3A with CycE–Cdk2 were generated using hs-FLP act>Gal4/UAS in pupal wings (F–H) and eyes (H). Clones were negatively marked by CD2 (F and G), and levels of GFP-tagged E2F1 were compared at 44 h APF. All GFP measurements were acquired at the same gain and at roughly similar tissue sections. Several samples (n = 7) across three independent experiments were compared for GFP intensity using ImageJ (National Institutes of Health), and representative examples are shown. Bars in H indicate the mean GFP intensity across all samples measured, and error bars indicate the standard deviation. GFP-E2F1^WT is destabilized in the presence of CycE–Cdk2 after exit in eyes and wings (F–H). Wings expressing either GFP-E2F1^WT or GFP-E2F1^PIP3A in the dorsal domain during pupal stages (under control of ap-Gal4/UAS, tub-Gal80^TS) were assayed for PH3. (I and J) No ectopic mitoses were evident in wings expressing either the stabilized or WT forms of E2F1 after 36 h APF (44 h APF shown). However, several ectopic ELAV⁺ neurons were found in the dorsal posterior margin of all GFP-E2F1^PIP3A–expressing wings (J, arrows). Bars, 50 µm.