repeat expression lines previously described expanded CAG,100, CTG,100 and CAA,100 constructs were subcloned into pBKS+ vector (GenBank/EMBL accession number X52327) and subse- quently inserted into pBD1010 (kindly donated by Professor Barry Dickson) upstream of GFP using EcoRI and XhoI restriction sites. The expanded repeat together with GFP was then cloned into pUAST-attB [65] using EcoRI and XbaI restriction sites. The length of the repeats and the integrity of GFP were confirmed by DNA sequencing. Quantification of Tergite Disruption Newly eclosed adult females were scored for tergite disruption by examining the dorsal abdomen under a standard dissecting microscope. The phenotype was categorised on the scale: 1, like wild-type; 2, tergites mildly disrupted but not split; 3, at least one tergite split; 4, two or more tergites split. Counts from multiple crosses scored under identical conditions were pooled to give a final tally for each genotype. To compare the effect of modifiers on the tergite phenotype categories were pooled into 2 groups, those in category one or two, and those in category three or four. This represents those with a mild, or no phenotype, and those with a strong phenotype, and appeared to be the most robust way to determine if modification was significant. Statistical significance was determined using Fisher’s exact test (GraphPad Prism). This enabled direct comparison of different sized populations, and determined the probability that the distribution of progeny within categories differ between genotypes by chance alone. p = 0.05 was used as a cut off for significance. Images of the adult abdomen were taken using an Olympus SZX7 microscope fitted with a SZX-AS aperture. Images were captured using an Olympus ColourView IIIU Soft Imaging System camera and AnalysisRuler image acquisition software. Adobe Photoshop CS was used for image preparation. In situ Hybridisation of Frozen Sections Whole wandering third instar larvae were frozen in Tissue-TekH O.C.T.TM freezing medium and a Leica CM1900 cryostat was used to cut 10 mm sections. For each genotype multiple larvae were frozen per mould such that each section contained multiple animals. Prior to hybridisation sections were fixed 15 minutes in ice cold 4% paraformaldehyde in PBS, washed 3615 minutes in room temperature PBS and quickly rinsed in 100% ethanol. Slides were dried and hybridised for 2 hours, or overnight at 37uC in a humid chamber with 0.5 ng/ul fluorescent oligonucleotide probe in hybridisation buffer (46SSC, 0.2 g/mL dextran sulphate, 50% formamide, 0.25 mg/mL poly(A) RNA, 0.25mg/mL single stranded DNA, 0.1 M DTT, 0.56 Denhardt’s reagent). Slides were washed 2615 minutes in 26SSC, 2615 minutes in 0.5xSSC at 37uC, air-dried and mounted in VectashieldTM (Vector labs) with 1 ng/mL DAPI to visualise nuclei. Fluorescent microscopy for in situ hybridisation experiments was performed using a Zeiss Axioplan 2 microscope with 636 PlanApo objective. Images were captured using Axiovision 4.5 software with an Axiocam MRm camera. Further preparation of images was done using Axiovision, or Adobe Photoshop CS. Multiple nuclei from multiple sections, each containing multiple animals (n<10) were examined per genotype. Independent transgenic lines were examined for each repeat construct. Fluorescent Probes for in situ Hybridisation Experiments Cy3-CTG10: Cy3- CTGCTGCTGCTGCTGCTGCTGCTGCTGCTG. Cy3-CAG10: Cy3-CAGCAGCAGCAGCAGCAGCAGCAG- CAGCAG. CUG and CAG Repeat RNA Pathogenesis in Drosophila Cy3-TTG10: Cy3- TTGTTGTTGTTGTTGTTGTTGTTGTTGTTG. Cy3-GFP: Cy3-CCTTCACCCTCTCCACTGACA- GAAAATTTGTGCCC. Supporting Information Figure S1 Graphs show the proportion of progeny within each category for all genotypes. Proportion (0.0 to 1.0) is shown on the y-axis while each category (1–4) is shown on the x-axis. Total population size, n, is indicated above each graph. A – K, phenotype when each line is ubiquitously expressed with da-GAL4. A’ – H’, phenotype when the same lines are crossed to w1118 to give progeny with all four repeat transgenes, in the absence of GAL4 driven expression. A, A’ w1118 wild-type lines. B, B’ 4xUAS control line. C, C’ 4xrCAA,100 [line 1] and D, D’ 4xrCAA,100 [line 2]. E, E’ 4xrCUG,100 [line 1] and F, F’ 4xrCUG,100 [line 2]. G, G’ 4xrCAG,100 [line 1] and H, H’ 4xrCAG,100 [line 2]. I, 2xrCAG,100 [line 1A] and J, 2xrCAG,100 [line 1B], the two transgene copy lines that were used to create 4xrCAG,100 [line 1]. When each of the two copies is expressed via da-GAL4, I, J, the resultant phenotype is weaker than in the 4 copy line, G. (TIF) Figure S2 Complete data sets showing proportion of progeny within each tergite phenotype category when repeat constructs were expressed in histoblasts with T155–GAL4. Population size, n, is shown above each graph. Significance indicated is based on comparing each repeat expression line to the EV control, using Fisher’s exact test to compare the distribution of progeny between those with any phenotype (category 2, 3 and 4) and those like wild-type (category 1). *p,0.05 and ***p,0.001. Only 4xrCAG,100 expression in, F, G, gives a significant phenotype. (TIF) Figure S3 Effect of reducing Mbl levels on the tergite phenotype. Each repeat line was expressed ubiquitously via da- GAL4 and via da-GAL4 in the presence of one copy of the mblE27 allele. A, B, C, expression of independent lines for each repeat construct, either in a Mbl wild-type background (left column), or in the presence of one copy of the mblE27 allele (right column). A, w1118 control, and two independent 4xrCAA,100 lines, B, two independent 4xrCUG,100 lines and C, two independent 4xrCAG,100 lines. D–G, statistical comparison (Fisher’s exact test) of the proportion of progeny with any phenotype (category 2, 3, 4) and a strong phenotype (category 3, 4) for D, 4xrCUG,100 [line 1], E, 4xrCUG,100 [line 2], F, 4xrCAG,100 [line 1] and G, 4xrCAG,100 [line 2]. *p,0.05, **p,0.01, ***p,0.001. (TIF) Figure S4 Effect of reducing Dcr-2 levels on the tergite phenotype. Each repeat line was expressed ubiquitously via da- GAL4 and via da-GAL4 in the presence of one copy of the dcr2L811fsX allele. A, B, C, expression of independent lines for each repeat construct, either in a Mbl wild-type background (left column), or in the presence of one copy of the mblE27 allele (right column). A, w1118 control, and two independent 4xrCAA,100 lines, B, two independent 4xrCUG,100 lines and C, two independent 4xrCAG,100 lines. D–G, statistical comparison (Fisher’s exact test) of the proportion of progeny with any phenotype (category 2, 3, 4) and a strong phenotype (category 3, 4) for D, 4xrCUG,100 [line 1], E, 4xrCUG,100 [line 2], F, 4xrCAG,100 [line 1] and G, 4xrCAG,100 [line 2]. *p,0.05, **p,0.01, ***p,0.001. (TIF) PLoS ONE | www.plosone.org 11 June 2012 | Volume 7 | Issue 6 | e38516