Regulation of Drosophila FKH/FOXA by TOR Figure 3. Quantitation of the cell size phenotypes shown in figure 2. Cell size was analyzed by measuring the cell perimeter with the software ImageJ. Blue bars represent the GFP-negative wild-type cells, red bars represent the cells expressing the transgene. Larvae were fed with yeast paste, starved on PBS or treated with 50 mM rapamycin. (A) Effect of FKH knockdown on cell size. Larvae were of the genotype y w hs-flp;; Act.CD2.Gal4 UAS-GFP/UAS-FKH-RNAi (bars labeled ‘‘FKH-RNAi’’) or y w hs-flp;; Act.CD2.Gal4 UAS-GFP/UAS-LacZ-RNAi (bars labeled ‘‘LacZ-RNAi’’) as an unspecific control. Cells expressing FKH dsRNA are significantly larger than the surrounding tissue in larvae which were starved on PBS or treated with rapamycin. (B) Effect of FKH overexpression on cell size. Larvae were of the genotype y w hs-flp;; Act.CD2.Gal4 UAS-GFP/UAS-FKH (bars labeled ‘‘UAS-FKH’’) or y w hs-flp;; Act.CD2.Gal4 UAS-GFP/UAS-LacZ (bars labeled ‘‘UAS-LacZ’’) as an unspecific control. Cells which overexpress FKH are significantly smaller than the surrounding tissue in fed larvae, but not in larvae starved on PBS. They are also significantly smaller than wild-type cells in larvae treated with rapamycin. Significance was tested using an unpaired 2-tailed Student’s t-test. * = p,0.05; ** = p,0.01; *** = p,0.001. Error bars represent SEM. Because the larvae are not age-matched across the different conditions (see Materials and Methods), we base our statements on the comparison of transgene-expressing cells to the wild-type control cells within the same sample, and not on the comparison of absolute cell sizes across different conditions. doi:10.1371/journal.pone.0015171.g003 regulation of CG6770 by FKH with a cell culture-based reporter gene experiment. When a 880 bp PCR product encompassing the CG6770 promoter was cloned upstream of a luciferase ORF, reporter gene induction could be achieved from the resulting plasmid by co-expression of FKH to a greater extent than by dFOXO (Figure S3). We are aware of the fact that these observations do not exclude the possibility that cabut and CG6770 are indirect FKH target genes, however we consider this of little relevance for their use as indicators for FKH activity. In a next step following the demonstration that both genes are induced by FKH, we addressed whether cabut and CG6770 transcription was also responsive to TOR signaling levels, and could thus be used as a readout to confirm the interaction between the TOR module and FKH. Rapamycin feeding elicited a robust induction of cabut PLoS ONE | www.plosone.org 5 December 2010 | Volume 5 | Issue 12 | e15171