Figure 5. N/Src synergy disrupts the cell cycle.
(A) DNA content analysis was performed on Hoechst-labeled dissociated cells from vgGal4;UAS-GFP wing discs expressing UAS-Src64B;UAS-Nact (dark green trace), UAS-Src64B (light blue), UAS-Nact (red), WT control (black), UAS-BskDN;UAS-Src64B;UAS-Nact (light green), UAS-Nact;UAS-Src42ACA (purple), UAS-Nact;d10338 (dark blue) or UAS- Nact;UAS-Mef2 (orange). Comparative histograms show relative frequencies on the y-axis, normalized to total number of counts for each sample. (B–E) EdU incorporation assay in dppGal4;UAS-GFP wing discs expressing d10338;UAS-Nact (B), d10338 (C), UAS-Nact (D), or UAS-GFP (E) at 22°C. A closeup of the areas denoted by boxes is shown below each image, and the GFP-positive area is marked with dotted yellow lines. Whereas UAS-Nact alone expands the ZNC (zone of non-proliferating cells) and also non-cell-autonomously induces proliferation in the dorsal-posterior region of the disc, thus increasing the size of the dorsal compartment (D), the combination of d10338 and UAS-Nact eliminates the expansion of the non-proliferative zone and causes cells within the ZNC proper to begin incorporating EdU; furthermore, the area of increased proliferation in the dorsal compartment appears to be expanded (B). (F–J) Nact and Src42ACA together cause a reduction in dacapo (dap) levels. (F) qPCR for dap expression in wing discs expressing Nact and/or Src42ACA or Mef2 under the vgGal4 driver. (G–J) A dap-LacZ reporter assay was used to visualize dap expression in vgGal4 wing discs in a dapk07309/+ background. Both Nact and Src42ACA together (G) and Src42ACA alone (H) show a reduction in dap-LacZ compared to both Nact alone (I) and vgGal4 controls (J). Scale bars: 100 μM.
Figure 5—figure supplement 1. Elimination of G1 phase of the cell cycle also occurs in dppGal4 wing discs expressing Nact and Src64B.
