TRIM50 Promotes Aggresome Formation and Clearance Figure 6. TRIM50-related aggresome and TRIM50 itself are partially degraded through the autophagy-lysosomal pathway. (A) FLAG- TRIM50#3 cells were treated with 20 mM NH4Cl (A) and DMSO for a period of 10 hours. Cells were lysed at the indicated time after the initiation of treatment and analysed by Western blotted using FLAG and GAPDH antibodies, respectively. The experiments were performed three times and typical results are shown. (B) Curves describing TRIM50 levels as a function of time, based on the results in panel A. The density of each band was determined by densitometer. The half-time of TRIM50 was determined by calculating the protein level at each time, normalized to the corresponding GAPDH level, to the initial amount of TRIM50 protein and compared with the control cells treated with DMSO. (C) HeLa cells overexpressing EGFP- TRIM50 were incubated in complete medium (a–c), EBSS (d–f), and DMEM supplemented with NH4Cl 20 mM for 2 h respectively and stained with an anti-FK2 antibody. doi:10.1371/journal.pone.0040440.g006 conjugated anti-mouse and anti-rabbit antibodies (GE Healthcare) and the ECL chemiluminescence system (GE Healthcare) was used for detection. Where indicated the MG132 proteasome inhibitor (Calbiochem, USA) was added. GST-p62 (231–385) and GST-p62 (232–370) fusion proteins were purified using glutathi- one-Sepharose 4B beads (GE Healthcare) according to the manufacturer’s instructions. For the GST pulldown assay, 3 mg of GST-recombinant proteins were mixed with 40 mg of total FLAG-TRIM50#3 cell lysate and incubated at 4uC for 2 h with rotation, and then incubated with FLAG antibody for 4 h. The binding fraction was washed four times and then loaded into a SDS 10% PAGE gel, and immunoblotted with anti-GST antibody (Santa Cruz). Soluble and insoluble fractions were obtained using RIPA buffer as described elsewhere (Muqit et al., 2006). Protein band densities were determined using densitometer (Kodak). The amount of the protein was calculated by the initial amount of FLAG-protein level and normalized with GAPDH. Cells treated with DMSO were used as control. Supporting Information Figure S1 TRIM50 does not colocalize with known and induced cellular organelles. (A) HeLa cells were transiently transfected with EGFP-TRIM50, fixed and stained for different cytoplasmic markers. The panel shows the merge of EGFP- TRIM50 (green) with single marker (red): Golgi markers (a,b,c,d,i), early endosomes (e), the coat complex of endoplasmic reticulum associated vesicles (f,g,h), lysosomes (j), cytoskeletal structures (k), and peroxisomes (l). (B) HeLa cells were transiently transfected with EGFP-TRIM50. After 24h the cells were cultured in presence of arsenite (0.5 mM for 30 min.) before processed and immuno- stained with anti-Eif3 for stress granules (b,c), and anti-HuR for P- bodies (e,f). (TIF) Figure S2 Movement of TRIM50 particles and determi- nation of the movement rates. (A) HeLa cells transiently expressing EGFP-TRIM50 were imaged over 110 frames at 0.5 sec intervals. The individual frames were summed using ImageJ. The tracks of three single particles are shown (blue, particle a; green, particle b; red, particle c). The asterisks indicate starting point of each particle. (B) Velocities of particles a–c were measured throughout the time that they were observed during the frames by using manual tracking plugin of ImageJ program. The vertical Y-axis shows the velocity of particles in each frame (micron/sec); the horizontal X-axis shows the relative time during the image sequence in which the particle was observed. (C) Recovery of the signal was measured throughout the time observed during 221 frames sequence using manual tracking of ImageJ program. (D) HeLa cells were transfected with EGFP- TRIM50, incubated for 30 min with TRITC-dextran, and imaged using confocal fluorescence microscopy. EM immuno- gold-labeled of a section corresponding to the area indicated by the box in figure D. (TIF) Figure S3 TRIM50 interacts with beta tubulin. (A) Endogenous TRIM50 colocalizes with polyubiquitinated proteins. SH-SY5Y cells were stained with anti-TRIM50 antibody and with FK2 antibody. Where indicated, the cells were treated with 25 mM MG132 for 6 h. (B) HeLa cells expressing EGFP-TRIM50 were stained with an anti-HDAC6 antibody. The cells were treated with 25 mM MG132 for 6 h (d–f). (C) The interaction between TRIM50 and beta tubulin was assayed in HEK293 cells transiently expressing FLAG-Tubb2b and EGFP-TRIM50. The cell lysates were immunoprecipitated with anti-FLAG and immunoblotted with anti-GFP antibody. (TIF) Figure S4 (A) TRIM50 E3-ubiquitin ligase activity is not required for aggresome localization. MEF Trim502/2 cell line was transfected with EGFP-Trim50DRING, treated with 25 mM MG132 for 6 h and stained with HDAC6 antibody (a–f). (B) TRIM50 colocalizes with LC3. HeLa cells were transiently co- transfected with HA-TRIM50 and DsRed-LC3 and stained with anti-HA antibody. After 24h the cells were incubated with EBSS medium for 2 h (d–f). (TIF) Figure S5 TRIM50 increases the insolubility of p62 and HDAC6 into the aggresome. (A–B–C) TRIM50 promotes the sequestration of p62 and HDAC6 in aggresome. Lysates from FLAG-TRIM50#3 and FLAG#3 cell lines, treated with vehicle (–) or with MG132 (+) were separated in detergent-soluble and detergent insoluble fractions and immunoblotting with anti-FLAG and anti-p62 antibodies. An example for p62 protein is depicted (A). The asterisk shows the relative band of endogenous p62 of previous immunoblotting. The relative level of soluble and insoluble fractions was measured by quantification of the intensity of p62 (B) and HDAC6 (C) bands of three indipendent experiments. (TIF) Movie S1 EGFP-TRIM50 bodies movement. Movies show a 110 frames sequence captured at 0.5 second intervals. (MOV) Movie S2 FRAP analysis of TRIM50 cytoplasmic bodies. Movies show a 224 frames sequence captured at 0.5 second intervals. (MOV) Plasmids used in this study. Table S1 (DOC) Table S2 TRIM50-associated proteins found ubiquiti- nated and/or present within induced Aggresome. (DOC) Acknowledgments We thank T. Johansen, M.W. Wooten and P.Matthias for plasmids and Joo-Yong Lee for providing with Hdac6 mouse fibroblast cell lines; we are grateful to Nicola Brunetti Pierri for careful reading of the manuscript. PLoS ONE | www.plosone.org 12 July 2012 | Volume 7 | Issue 7 | e40440