Studying Anticancer Drug Properties in Drosophila Figure 6. The Drosophila white-mottled inversion: a classical system to study long-range regulation of gene expression. (A) Schematic representation of the Drosophila X chromosome and of the white-mottled (wm4h) inversion that juxtaposes the white gene to a heterochromatin context, in this case within the 4-Mbp rDNA locus adjacent to the SAT III array. Different features of the proximal end of the chromosome, most of which remains unsequenced, are discussed in the text. Stochastic silencing of white leads to a mottled eye color (bottom left corner) compared to the wild-type red adult eye (top right corner). This phenomenon corresponds to the classical definition of position-effect variegation (PEV). The nearby Notch gene is similarly affected under certain growth conditions (see text for details). (B) Intrinsic loss of symmetry resulting from white and Notch PEV in white-mottled flies. The fly shown is a representative example of an adult phenotype observed at 1–2% frequency in the wm4h line when grown at 18uC. The facing view illustrates a characteristic mottled color of the left eye, but a wild-type red color on the right. This is more clearly seen in the right and left close-ups shown to the right. This intrinsic propensity for asymmetry in wm4h PEV manifestations most likely results from Notch misregulation and is important to understand the one-sided phenomena illustrated in Figure 8. doi:10.1371/journal.pone.0023597.g006 polish and the samples photographed under UV illumination using a Leica DMRB microscope equipped with a CoolSnap HQ CCD camera. Photographs were processed with Adobe Photoshop to render grey scale images in false colors and corrected for black balance. In vivo drug-feeding experiments (‘‘oral administration’’) The chief characteristics of the white-mottled (wm4h) line used in these experiments are illustrated in Figure 6. Batches of 30 late third-instar L3 wm4h or wild-type Oregon R larvae were transferred to agar plates containing 0, 10, 25, 50 or 100 mM etoposide or teniposide or with 1, 5, 25, 50 or 100 mM F14512 and yeast paste supplemented with the same concentration of the drugs. Late L3 larvae feed actively and can be easily picked from a Drosophila culture as they are the most mobile. This insures that the larvae will fairly uniformly eat the drug-laced yeast paste, the drug present in the agar preventing dilution. Treatment with 100 mM teniposide or 50 mM F14512 led to .90% lethality. No lethality (,10%) was observed following treatment with lower concentrations of the drugs (up to 75 mM teniposide and 25 mM F14512), which led, however to developmental delays during pupation. Eye pigment levels (Figure 7) were measured from the heads of 30 5-day old adult female flies after photography as previously described [36] or, in the case of F14512-fed flies, from 10 female heads as drug concentrations of 25 or 50 mM lead to approximately 50-70% lethality. Males were examined as early as 1 or 2 days following hatching, as Eye wide shut adults die within 2 days. This provides enough time to score for the gain- or loss-of-function phenotypes shown in Figure 8. Flies were PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e23597