TRIM50 Promotes Aggresome Formation and Clearance Figure 5. TRIM50 promotes the accumulation of polyubiquitinated proteins into the aggresome. (A) MEF Trim50 cell lines were stained with anti-FK2 antibody. The area, perimeter and intensity of signal of three independent experiments of each genotype were estimated using imageJ program. (B) MEF Trim50 cell lines were treated with 10 mM MG132 over night and stained with anti-FK2 antibody. The area, perimeter and intensity of signal of three independent experiments of each genotype were estimated using imageJ program. (C) MEF Trim50 cell lines were treated with 10 mM MG132 over night, incubated o and 48 h with DMEM after MG132 wash out and stained with anti-FK2 antibody. The diagram shows the percentage of aggresome-positive cells. doi:10.1371/journal.pone.0040440.g005 heterozygote mice (F1) have around twenty ‘‘background’’ mutations in addition to the wanted one. They were backcrossed with C3HeB/FeJ wild-type mice from Jackson laboratories for 12 generations to purge the strain of other mutations potentially induced by the ENU treatment. Mouse embryonic fibroblasts (MEFs) were prepared from E13.5 embryos as described in [40]. Cells were subsequently genotyped by Sanger sequencing to identify +/+, +/2 and 2/2 lines. All procedures used with mice models and to generate the MEFs were approved by the CIG Institutional Animal Care and followed the National Institutes of Health Guidelines, ‘Using Animals in Intramural Research’. The work was approved by the ethics committee of the Veterinarian Cantonal Office (authorization Vaud-1958). Immunofluorescence Microscopy For immunofluorescence analyses, the cells transfected with EGFP-TRIM50 were fixed before their incubation with the primary and secondary antibodies of interest, mounted in mowiol and examined on a Zeiss LSM 510 META confocal microscope (Carl Zeiss, Jena, Germany). All confocal images were obtained using the necessary filter sets for GFP, Alexafluor 488 and 546, using a Zeiss Plan-Neofluor 636 oil immersion objective (NA 1.4), with the pinhole set to one Airey unit. Live Cell Imaging and Fluorescent Recovery After Photobleaching (FRAP) Analyses HeLa cells were transfected with EGFP-TRIM50 construct and observed at 37uC in 20mM HEPES buffered DMEM using a Zeiss LSM 510 META confocal microscope (Carl Zeiss, Jena, Germany). Temperature was controlled with a Nevtek air stream stage incubator (Burnsville, VA, USA) and images of live cells were acquired using sequential excitation at 488 nm and 543 nm. The tracking of moving objects and evaluation of their speed were performed using the Tracking macro of the ImageJ program. Selective photobleaching in the regions of interest within the cell was carried out on the Zeiss LSM510 using 100 iterations with a 488 nm laser line at full power. Correlative Light Electron Microscopy (CLEM) EGFP-TRIM50 transfected HeLa cells were grown on CELLocate coverslips with coordinated grid and then prepared for CLEM microscopy identification of TRIM50 structures according to Polishchuk et al. [41]. Briefly, after visualization of EGFP-TRIM50 positive bodies by time-lapse confocal microsco- py, the cells were fixed, labeled with an antibody against EGFP using the gold-enhance protocol, embedded in Epon-812, and cut in serial sections. Then region containing TRIM50-positive bodies were analyzed in serial thin sections under a Philips Tecnai-12 electron microscope (Philips, Einhoven, The Netherlands). EM images were acquired from the region of interest using an Ultra View CCD digital camera (Soft Imaging Systems, Munich, Germany). Statistical Analysis All microscopy experiments were performed in triplicate. Approximately 40 cells were analyzed for each experimental condition. For the immunofluorescence experiments on Trim50- MEF cells, area, perimeter, and intensity of FK2-positive dots were measured using ImageJ program. The threshold was set to a level that excludes all the cytosolic background, thus allowing selective analysis of intense puncta representing the FK2-positive dots. Data were reported as median along with the upper and lower quartiles (Q1–Q3). Normal distribution assumption was checked by means of Q-Q plot, Shapiro-Wilks and Kolmogorov- Smirnov tests. The followed parameters were log transformed before statistical analyses because of their skewed distribution. Comparisons between wild-type, heterozygous and knockout mice were assessed by means of specific contrasts defined into a hierarchical linear model (HLM), accounting for clustering due to multiple measures collected within cell. All p-values were adjusted for multiple comparison following Tukey-Kramer’s method. A p- value ,0.05 was considered for statistical significance. All statistical analyses and graphs were performed using SAS Release 9.1 (SAS Institute, Cary, NC, USA) and R (version 2.10.1) software, respectively. Protein Identification by Mass Spectrometry Analysis TRIM50 complexes were isolated from HEK293 cells total extracts by immunoprecipitation. FLAG-TRIM50#3 and FLAG#3 cell lines were lysed in PBS, 0.5% NP-40, 1 mM PMSF, and COMPLETE protease inhibitors (Roche) for 45 min under gently mixed. Total protein extracts were pre-cleared with unspecific Mouse IgG Agarose Beads (Sigma) overnight in lysis buffer. The protein extracts were recovered by centrifugation (3000 rpm for 5 min) and then incubated overnight, under gently agitation, onto M2 anti-FLAG agarose-conjugated antibody beads (Sigma) previously blocked with no fat milk treatment. Unbound proteins were discarded and the beads were collected by centrifugation and extensively washed with lysis buffer supple- mented with 150 mM NaCl to eliminate non-specific bound proteins. Elution of the desired protein complexes was performed by competition with FLAG peptide in elution buffer. The eluted proteins were precipitated in methanol/chloroform and then loaded onto a 10% SDS-PAGE. The gel was stained with colloidal Coomassie blue (Pierce). Protein bands were excised from the gel, reduced, alkylated and digested with trypsin as described elsewhere (Zito et al., 2007). Peptide mixtures extracted from the gel were analyzed by nano-chromatography tandem mass spectrometry (nanoLC–MS/MS) on a CHIP MS Ion Trap XCT Ultra equipped with a capillary 1100 HPLC system and a chip cube (Agilent Technologies, Palo Alto, CA). Peptide analysis was performed using data-dependent acquisition of one MS scan (mass range from 400 to 2000 m/z) followed by MS/MS scans of the three most abundant ions in each MS scan. Raw data from nanoLC–MS/MS analyses were employed to query a non- redundant protein database using in house MASCOT software (Matrix Science, Boston, USA). Immunoprecipitation, GST Pull-down and Western Blot Co-immunoprecipitation experiments were performed as pre- viously described [42]. Complexes were analyzed by western blotting using indicated antibodies. Horseradish peroxidase PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e40440