Figure 7. Transient siRNA-mediated knockdown of TRAP1 increases [A53T]a-Synuclein-induced mitochondrial fragmen- tation. SH-SY5Y cells were co-transfected with the siRNAs and plasmids indicated. Mitochondria were visualized by DsRed targeted to mitochondria (mito-DsRed). (A) Confocal images taken of representative cells displaying either an intact tubular mitochondrial network (control siRNA) or a fragmentation of the network (control siRNA+A53T). Transient knockdown of TRAP1 causes mitochondrial fragmentation itself (TRAP1 siRNA), additional co-expression of [A53T]a-Synuclein aggravated this phenotype and led to an overall increase in cells showing a fragmented mitochondrial network (TRAP1 siRNA+A53T). (B) For quantification, at least 300 transfected cells per coverslip were TRAP1 Mitigates a-Synuclein Toxicity analyzed. The mitochondrial morphology was determined in a blinded manner. Quantifications are based on triplicates of three independent experiments. (C) Expression levels of [A53T]a-Synuclein and TRAP1 were analyzed by Western Blotting. b-Actin was used as a loading control. **p,0.01; ***p,0.001 (ANOVA). doi:10.1371/journal.pgen.1002488.g007 Center (VDRC) or from National Institute of Genetics (NIG-fly, Japan). Bloomington lines used were: Wild type flies (Oregon R; referred to as + in text), w1118;; P{Ddc-GAL4.L}4.36 (BL7009; ddc: dopa decarboxylase, aminergic neuron specific driver, referred to in text as ddc-GAL4), w*; P{UAS-lacZ.B}Bg4-2-4b (BL1777; referred to in text as UAS-LacZ), y1w67c23; P{SUPor-P}Trap1KG06242 (BL14032; referred to in text as TRAP1[KG]), w[*]; P{w[+mC] = longGMR-GAL4} (BL8605; referred to as GMR- GAL4 in text), w[1118]; P{w[+mC] = UAS-HsapHSPA1L.W}53.1 (BL7455; expresses HSPA1L, the human homolog of HSP70 under GAL4 control; referred to as HSP70 in text), w[1118]; P{w[+mC] = UAS-mitoGFP.AP} (BL8443; expresses GFP with a mitochondrial import signal; referred to as mito-GFP in text) and w[*]; P{w[+mC] = UAS-Hsap\MJD.tr-Q78}c211.2 (BL8150; ex- presses a HA-tagged C-terminal fragment of the human Machado-Joseph Disease/Spinocerebellar Ataxia 3 protein with a 78 repeat polyglutamine tract; referred to in text as PolyQ). For stable eye-specific expression of PolyQ, GMR-GAL4 driver was recombined with PolyQ transgene (GMR.PolyQ in text). Fly lines suitable for GFP (yw;; UAS-GFP) or human [A53T]a- Synuclein (yw;; UAS-[A53T]a-Synuclein [41]) expression (referred to in text as GFP or A53T, respectively). Stable expression under control of the ddc driver (w[*];; ddc-GAL4.UAS-GFP; w[*];; ddc- GAL4.UAS-GFP) were generated by recombination (flies referred to in text as ddc.GFP or ddc.A53T). Transgenic flies expressing human TRAP1 (hTRAP1) were generated by BestGene Inc. In brief, human TRAP1 cDNA was sub-cloned from pcDNA3.1+ vector into pUASattB using KpnI and XbaI restriction sites. hTRAP1 expression in these transgenic flies (w;; UAS-hTRAP1/TM3, Sb; referred to in text as hTRAP1) was verified by Western blotting. Genetic Deficiency Screen Breeding The Bloomington Deficiency Kit was utilized for screening purposes (http://flystocks.bio.indiana.edu/Browse/df/dfkit_retir- ed_July2009.htm). In general, a specific deficiency line was crossbred with ddc.A53T flies. Male offspring (ddc.A53T flies in combination with the respective deficiency) was selected and aged. At ages 1 and 4 weeks, a minimum of 9 flies was collected for later measurement of head DA using HPLC. Measurement of Fly Head Dopamine (DA) using HPLC Liquid nitrogen flash frozen fly heads were homogenized (Precellys 24 homogenizer) in homogenization buffer (0.1 M perchloric acid/3% trichloric acid solution). 50 ml of supernatant from each sample were used for HPLC analysis (Dionex Ultimate 3000; running buffer: 57 mM citric acid, 43 mM sodium acetate, 0.1 mM EDTA, 1 mM octane sulfonic acid, 20% methanol). Samples were separated on a chromatographic column (Dionex Acclaim C18, 5 mm, 2.16150 mm column, at 25uC), and DA was electrochemically detected on a graphite electrode (Dionex ED50 Electrochemical detector with following conditions: disposable carbon electrode at 0.8 V, flow rate 0.2 ml/min). DA (Sigma- Aldrich) standards of 0.1 mM, 0.25 mM and 0.4 mM were used for creation of a standard curve. Chromeleon 6.6 software was used for HPLC data analysis. PLoS Genetics | www.plosgenetics.org 10 February 2012 | Volume 8 | Issue 2 | e1002488