or KAT60c were lysed in 1 ml ice cold lysis buffer (50 mM Tris-Cl pH 7.6, 150 mM NaCl, 10% glycerol, 0.1% NP40, 1 mM EDTA, 1 mM EGTA) containing protease inhibitors (234 mM leupeptin, 500 mM phenylmethanesulphonylfluoride (PMSF), 7.3 mM pep- statin A, 2.5 mM pefabloc, 250 mM 1,10 phenanthroline and 10 mM E-64d) and incubated for 15 minute on ice. Cell debris was removed by centrifuging at 100,0006g at 4uC for 45 minutes. The resulting supernatant was incubated with 100 ml anti-HA affinity matrix (Roche) for 2 hours at 4uC to immunoprecipitate 3xha:GFP:KAT80. Following 4 washes with 1 ml of wash buffer (50 mM Tris-Cl pH 7.6, 150 mM NaCl, 10% glycerol, 0.1% NP40), immunoprecipitated proteins were eluted by boiling in Laemmli buffer. Eluted proteins were analysed by SDS-PAGE followed by Western blotting probing with antibodies against the tags (as above). Electron microscopy Specimens were prepared for electron microscopy by Laurence Tetley and Margaret Mullin, Integrated Microscopy Facility, University of Glasgow. For scanning electron microscopy (SEM) analyses, approximately 107 bloodstream stage T. brucei cells were centrifuged at 15006g for 5 minutes and washed once in Trypanosome Dilution Buffer (TDB,[87]) before being resus- pended in 100 ml TDB and then fixed in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer (SC) for 1 hour. Cells were washed 3 times in 0.1M SC, before a droplet of concentrated cell suspension in 0.1M SC was placed onto a 10 mm diameter poly- L-lysine coated glass coverslip and cells allowed to settle onto the coverslip for 30 minutes. Following a brief rinse in 0.1M SC to remove unattached cells, coverslips were incubated in 1% osmium tetroxide in 0.05 M SC for 1 hour. Coverslips were then washed 3 times in dH2O before being incubated with 0.5% uranyl acetate in the dark for 1 hour. Uranyl acetate was removed by 2 washes with dH2O and cells were dehydrated in an alcohol series before being subjected to critical point drying for 1 hour 20 minutes. Specimens were then mounted on aluminium stubs using double sided copper tape and coated with gold/ palladium in a Polaron SC515 sputter coater prior to examina- tion using a JEOL 6400 scanning electron microscope operating at 6 kV. Images were captured using an ADDA3 digital capture system (Olympus/SIS, Germany). For transmission electron microscopy (TEM), a pellet of approximately 107 T. brucei bloodstream stage cells was fixed in 2.5% glutaraldehyde in 0.1M SC for 1 hour. Following 3 washes in 0.1M SC, the fixed pellet was incubated with 1% osmium tetroxide/0.05M SC for 1 hour, washed 3 times in dH2O and then incubated in 0.5% uranyl acetate in the dark for 1 hour. Following 2 rinses in dH2O, the cell pellet was dehydrated in an alcohol series, incubated in 3 changes of propylene oxide, before being incubated overnight in a 50:50 mix of propylene oxide/ TAAB araldite 502/512 resin. The propylene oxide was then allowed to evaporate to leave pure resin, which was changed twice before the pellet was embedded in fresh resin, which was allowed to polymerise at 60uC for 48 hours prior to cutting 60– 70 nm thick sections and contrast staining with 2% uranyl acetate in methanol for 5 minutes and Reynolds’ lead citrate for 5 minutes. Supporting Information Figure S1 KAT80 is important for normal proliferation and cytokinesis in procyclic T. brucei. A. Cumulative growth curves of two independent procyclic KAT80 RNAi clones. Katanins and Spastin in Trypanosome Cytokinesis B. Western blotting showing downregulation of KAT80 expression following RNAi induction. One allele of KAT80 was replaced with an epitope tagged copy of the gene (3xha:GFP:KAT80) in each of the KAT80 RNAi cell lines in (A). Whole cell lysates (5 days post- induction with tetracycline (tet)) were analysed by Western blotting with anti-HA antibody (top panels). Portions of the Western membrane, stained with Ponceau after transfer (lower panels), are included as loading controls. C. DAPI staining. Following RNAi induction, cell cycle progression was monitored by DAPI staining at the time points indicated in days (d) (n.200). The nucleus (N) and kinetoplast (K) configurations of cells are given. ‘Others’ represents multinucleate/multikinetoplast cells. Data presented are for clone 1 and are representative of data for clone 2 (not shown). D. DNA content analysis. The fluorescence of 10,000 propidium iodide-stained cells was analysed by flow cytometry in the FL2-A channel at the time points (in days) indicated. The ploidies of the peaks are given. (TIF) Figure S2 TEM analysis of microtubule spacing follow- ing induction of KAT80 RNAi. Transverse cross-sections of cells plus or minus induction with tetracycline (tet) for 12.5 hours are shown. Regions indicated by the black boxes are enlarged 3 fold to visualise the subpellicular microtubules. Scale bars: 1 mm. (TIF) Figure S3 Cytokinesis stage analysis of 2N2K cells following depletion of KAT60a, KAT60b or KAT60c. A– C show data for KAT60a, KAT60b and KAT60c, respectively. n.100 cells per time point. Error bars represent the standard deviation of 3 biological replicates. (TIF) Figure S4 TEM analysis of microtubule spacing follow- ing induction of SPA RNAi. Transverse cross-sections of cells plus or minus induction with tetracycline (tet) for 12.5 hours are shown. Regions indicated by the black boxes are enlarged 3 fold to visualise the subpellicular microtubules. Scale bars: 500 nm. (TIF) Figure S5 3xha:GFP:KAT80 does not appear to interact with KAT60c:myc. Co-immunoprecipitation analysis of inter- actions between 3xha:GFP:KAT80 and KAT60c:myc. Anti-HA antibody was used to immunoprecipitate 3xha:GFP:KAT80 from cell lysates co-expressing KAT60c:myc, as shown by Western blot analysis with anti-HA antibody (top panels) of input (I), flow through (FT), first and last wash (W1 and W4, respectively) and elution (E) fractions. The input, flow through and elution fractions were then blotted with an anti-myc antibody to detect KAT60c:- myc. (TIF) Oligonucleotides used in this study. Table S1 (DOC) Acknowledgments We thank Margaret Mullin and Laurence Tetley for preparation of specimens for electron microscopy, Mike Turner for performing the statistical analyses and Keith Matthews for the gift of anti-TY antibody. Author Contributions Conceived and designed the experiments: CB CC JCM TCH. Performed the experiments: CB CC TCH. Analyzed the data: CB CC JCM TCH. Contributed reagents/materials/analysis tools: CB CC TCH. Wrote the paper: TCH. Commented on manuscript drafts: CB CC JCM. PLoS ONE | www.plosone.org 10 January 2012 | Volume 7 | Issue 1 | e30367