Fig. 5. dRheb expression correlates with S-phase during embryogenesis and promotes S-phase in S2 cells. In stage 13 embryos, the same domains express high levels of dRheb and incorporate BrdU into DNA: (A, left) in situ hybridization; (A, right) staining with anti-BrdU. At this stage, mitoses are taking place in the ventral nerve cord and brain (arrows), and endoreplication is occurring in the gut, including a central domain of the midgut (black arrowheads). To test the effects of overexpressing dRheb at the cellular level, S2 cells were transfected with pRmHa-GFP either alone or in conjunction with pPacFLAG-dRheb, and GFP expression was induced 16 hours later by addition of 500 µM CuSO₄. Cells were harvested 48 hours after induction and FACS was performed after gating for GFP-positive cells as described in Materials and Methods. Cells overexpressing dRheb exhibit an increased proportion of S phase (B); a small panel on the right summarizes the cell cycle profile of these cells. Forward scatter analysis of the same cells reveals that overexpression of dRheb results in an increase in cell size (C); quantitation of the forward scatter results is shown on the right. For FACS analysis of untransfected cells, 10,000 cells were collected and single cells were gated using an FL2-width versus FL2-area density plot for cell cycle progression. A gate using SSC-H versus FSC-H was used for cell size analysis. For FACS analysis of transfected cells, 5000-7000 GFP-positive cells were collected and cells were gated using FL1-H versus FL2-area density plot. Cell size was analysed using the SSC-H versus FSC-H gate. Mean FSC-H of the GFP-positive cell population was calculated from three independent experiments; error bars represent standard deviation from the mean.

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