Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo
1 Department of Biology, City College of the City University of New York, New York, USA
2 Department of Experimental Pathology, University of Bologna, Bologna, Italy
3 Institute of Molecular Systems Biology, ETH, Zürich, Switzerland
4 Courant Institute of Mathematical Science, New York University, New York, USA
5 Department of Genetics and Development Columbia University, New York, New York, USA
BMC Biology 2011, 9:65 doi:10.1186/1741-7007-9-65Published: 27 September 2011
Additional file 1:
Quantitative RT-PCR comparing the transcript levels for dm, cyclin D and cyclin E in Drosophila S2 cells upon insulin (A) or amino acids (B) treatment. Cells were treated with insulin or AAs and RNA was extracted at the indicated times. qRT-PCRs were performed to analyze expression of diminutive (dm), cyclin D and cyclin E RNAs. The sequences of the primers used are available in Additional file 8; Supplementary Material and Methods. actin was used as the internal control. Error bars indicate the standard deviations (±) calculated on the average of three separate experiments.
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Additional file 2:
Quantitative RT-PCR of dm and its target fibrillarin in Drosophila S2 cells upon insulin treatments and in the presence of rapamycin. Cells were treated with insulin or rapamycin alone and together as indicated in the figure; rp49 (ribosomal protein 49) was used as internal control. Similar results were obtained using actin as a control (not shown). Error bars indicate the standard deviation (±) calculated from three independent experiments.
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Additional file 3:
Appendix 1. Significance analysis of the conjectured growth for ommatidial number and size used in the experiments outlined in Figure 4 and Table 1. The P-values of a two-tailed z test for the analysis are represented in Table S1 in Additional file 4.
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Additional file 4:
Table S1. Representation of the relative increase of the size for each ommatidium and their total number. The values represent the percentage of increase in size (a-Size) or number (a-Number) as compared to their control and relative genetic background (see also data in Table 1). da is the standard deviations and the total number of animals used is indicated in Table 1. To establish the significance of the relative differences of the data within the different dm genetic background, we calculate the P-values using a standard two-sided z-test (the formula used is represented in Appendix 1 of Additional file 3). * complete genotype: the construct tubulin-FRT-dmyc-cDNA-FRT-Gal4, ey-Flp/Y was recombined into the dm+, dmpoor dm4 genetic background.
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Additional file 5:
Table S2. Analysis of the ommatidial size and number in animals with different dm genetic background. The total number of ommatidia and the relative size of each ommatidium are indicated. Standard deviations (±) are calculated based on the total number of the animals reported in parenthesis. Values represent the relative increase in the size (a) or number (b) of the ommatidia compared to the values in their genetic background (100). P-values are calculated from Student t test and are reported for the calculation of ommatidia number (c) and size (d). * complete genotype: the construct tubulin-FRT-dmyc-cDNA-FRT-Gal4, ey-Flp/Y was recombined into the dm+, dmpoor dm4 genetic background.
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Additional file 6:
BrdU-labeling in the eye imaginal discs from third instar larvae expressing RhebAV4 transgene in wild-type dm+/Y (A) or in hypomorphic dmP0/Y animals (B). Expression of the UAS-RhebAV4 did not significantly alter the S phase in the cells of the eye imaginal disc, visualized by BrdU labeling (red). Nuclei are labeled with DAPI (blue). Posterior is to the left.
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Additional file 7:
Expression of RhebAV4 induces Myc-dependent apoptosis. Third instar eye imaginal discs from ey-dm+/Y or ey-dmP0/Y larvae carrying the UAS-RhebAV4 transgene (A-B) or control chromosome (C-D) were tested for the presence of apoptotic cells. Discs were stained with anti-active caspase 3 antibody (red) to visualize cell death, or with anti ELAV (green) to mark the differentiated neuronal cells posterior to the morphogenetic furrow (MF). DAPI staining (blue) indicates nuclei. (E) Quantification of caspase-positive cells in the region posterior to the MF of the indicated genotype (visible in insets). Error bars indicate standard deviation (±) calculated from six independent eye imaginal discs. P < 0.001for t test for ey- dm+/Y; UAS-RhebAV4 vs. ey- dmP0/Y; UAS-RhebAV4 while comparisons within the other genotypes resulted in P > 0.1. (F) Photo of an eye imaginal disc from third-instar ey- dm+/Y; UAS-GFP larvae highlighting the territory where the eyeless-Gal4; UAS-GFP transgene is expressed. Posterior is to the left.
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Additional file 8:
MARCM showing a partial rescue of Rheb7A1 hypomorphic mutant clones by Myc overexpression. Rheb7A1 homozygous mutant clones suffer of growth disadvantage . These clones, induced at 72 h AEL and marked by GFP expression, are significantly smaller than wild-type siblings, which are marked by CD2 staining (red). The growth defect of Rheb7A1 mutant clones (A) is partially rescued by expression of Myc (B). Those clones are visible by co-expression with GFP, while wild-type clones are marked by the expression of CD2 and visualized by immunofluorescence using anti-CD2 antibodies. To generate MARCM clones the line hs-flp, tub-Gal4 (w+), UAS-GFP (w+); FRT82 [hsCD2 (y+)] tub-Gal80 was crossed with the line w; FRT82 Rheb7E1/TM6b (A) or with w; UAS-dMyc; FRT82 Rheb7E1/TM6b (B). (see Supplementary Material and Methods in Additional file 9).
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