PMC

A–E) Detection of DNA damage by anti-phospho-H2Av staining (red) in GMR-Gal4 third instar larval eye imaginal discs expressing GFP or the indicated GFP-E2f1 fusion proteins (green). Arrowheads indicate the position of the MF, with anterior to the bottom and posterior to the top. Bars = 10 µM. F) Quantification of the number of phospho-H2Av positive cells posterior to the MF. * p<0.001 relative to UAS-E2f1 expression.

A) Schematic of the E2f1^336–805 mutant protein, which contains an NH₂-terminal HA tag. B) Detection of apoptosis via Cleaved Caspase-3 (CC3, red) staining of third instar larval wing imaginal discs expressing HA-E2f1^336–805 (anti-HA, green) with en-Gal4. Bar = 50 µm. C) Anti-E2f1 western blot of third instar imaginal wing discs expressing GFP-E2f1, GFP-E2f1^Stable, or HA-E2f1^336–805. D) Co-immunoprecipitation analysis of Myc-E2f1 and HA-Rbf1 from transiently transfected S2 cells.

A–C) Detection of GFP or the indicated GFP-E2f1 proteins (green) in en-Gal4, UAS-p35 wing discs stained with DAPI (white). Scale bars indicate 50 µm. D) Quantification of morphological defects by microscopically measuring the thickness of the posterior compartment of the indicated en-Gal4>GFP-E2f1 wing discs. Measurements were obtained by counting the number of 1 micron sections required to visualize all the way through the posterior compartment of the tissue. Bars = 50 µM. E) E2f1^Stable induces apoptosis in two ways.

A–E) Detection of S phase by EdU labeling (red) and apoptosis by CC3 staining (green) in GMR-Gal4 third instar larval eye imaginal discs expressing GFP or the indicated GFP-E2f1 fusion proteins. Arrowheads indicate the position of the MF, with anterior to the left and posterior to the right. Bars = 5 µM. F) Quantification of the number of CC3 positive cells posterior to the MF. * p<0.001 relative to UAS-E2f1 expression.

A–I) Detection of apoptosis via Cleaved Caspase-3 (CC3, red) staining of third instar larval wing imaginal discs expressing the indicated GFP-E2f1 (GFP, green) proteins with en-Gal4. Arrow in D indicates an example of apoptosis observed in wild type wing discs. Bars = 50 µM. J) Quantification by flow cytometry of GFP-positive apoptotic cells from trypsin-dissociated en-Gal4 wing discs expressing GFP or the indicated GFP-E2f1 fusion proteins. Error bars represent the standard error of three independent experiments. ** p<0.01, * p<0.001.

A, B) qRT-PCR quantification of hid mRNA (A) or rpr mRNA (B) in en-Gal4 wing discs expressing GFP or the indicated GFP-E2f1 fusion proteins that lack (grey) or contain (black) the PIP3A mutation relative to a non-transgenic w¹¹¹⁸ control (Con). * p<0.001. C–I) Detection of apoptosis via Cleaved Caspase-3 (CC3, red) staining of third instar larval wing imaginal discs expressing the indicated GFP-E2f1 (GFP, green) proteins with en-Gal4. En-Gal4>E2f1^Stable (C–E) or E2f1^Stable/DBD (F–H) in either a wildtype hid background (+/+), or heterozygous Hid⁰⁵¹⁴¹/+ or Df(3L)H99/+ backgrounds. I) Quantification of CC3 pixel intensity as measured using ImageJ. All genotypes were normalized against E2f1^Stable; +/+ cleaved caspase-3 levels. * p<0.001. n.s. not significant. n = 12 discs for each genotype.

A) Schematic of the experimental paradigm. B) Schematic representation of E2f1 alleles used in this study. C) qRT-PCR quantification of GFP-containing mRNA in en-Gal4 wing discs expressing GFP or the indicated GFP-E2f1 fusion proteins that lack (grey; “N") or contain (black; “Y") the PIP-3A mutation () relative to a non-transgenic w¹¹¹⁸ control (Con). Error bars represent the standard error of three independent experiments. These designations will be used throughout the remaining figures. UAS-GFP expression was greater than any E2f1 construct because the UASt promoter was used rather than UASp. D) Anti-E2f1 western blot measuring GFP-E2f1 and endogenous E2f1 expression in third instar imaginal wing discs. The ratio of transgene expression to endogenous E2f1 expression is shown below. E) Quantification by flow cytometry of RFP-positive G1 cells from trypsin-dissociated en-Gal4, UAS-RFP wing discs expressing GFP or the indicated GFP-E2f1 fusion proteins. * p<0.001 as compared to GFP-E2f1 expression. F) qRT-PCR quantification of RnrS mRNA in en-Gal4 wing discs expressing GFP or the indicated GFP-E2f1 fusion proteins. G, H) Co-immunoprecipitation analysis of Myc-E2f1 and HA-Dp (G) or HA-Rbf1 (H) from transiently transfected S2 cells. I) Quantification by flow cytometry of GFP-positive S phase cells from trypsin-dissociated en-Gal4 wing discs expressing GFP or the indicated GFP-E2f1 fusion proteins. * p<0.001 compared between stabilized and normally degraded proteins.