Figure 7. TIG3 impact on organelle marker protein level. SCC-13 cells were infected by 10 MOI tAd5-EV or tAd5-TIG3 and after 24 h cell lysates were prepared for immunoblot detection of the indicated proteins. TIG3 expression reduces the level of rab11, GM130 and EEA1, but does not alter LAMP1 level. Molecular weights are indicated to the left of the blot in kDa. doi:10.1371/journal.pone.0023230.g007 distribution of TIG3 in normal human keratinocytes and these studies strongly suggest a pericentrosomal TIG3 location (not shown). Based on these findings, we argue that the major effect of TIG3 is at the centrosome and that the appearance of colocalization of TIG3 with organelle markers is an artifact due to pericentrosomal organelle clustering. Organelle relocation is a well-known phenomenon that occurs during apoptosis [45]. It is thought to enhance organelle membrane mixing to facilitate spread of pro-apoptotic effectors [45–50]. However, how these organelles and organelle fragments come together during apoptosis it is not well understood, but is thought to involve the centrosome and microtubules. The centrosome serves as a microtubule organizing center (MTOC) in interphase cells and as an organizer of the mitotic apparatus in mitotic cells. As a microtubule nucleation center, centrosome function is required for intracellular organelle trafficking [24,51]. Organelles move along microtubules associated with specific motor proteins [27,51]. Previous reports implicate microtubule motors in bringing organelle and organelle fragments to the microtubule organizing center (centrosome) during apoptosis [45,52]. These include the Golgi apparatus, endosomes, endo- plasmic reticulum, mitochondria and other organelles [45, 47,53,54]. The best characterized example is redistribution of mitochondria to the Golgi-proximal microtubule organizing center in cells exposed to TNFa, oxidative stress or viral infection [45]. Several reports suggest that this process activates MAPK signaling to phosphorylate kinesin light chain to halt mitochondria anterograde dispersal leading to accumulation of these organelles near the centrosome [45]. These previous reports describe pericentrosomal organelle clustering in response to treatment with growth factors, oxidative stress or other stimuli [45]. Our present studies are unique in that the organelle clustering is triggered by expression of a tumor suppressor protein. Moreover, the fact that TIG3 accumulates near the centrosome suggests that it may have a direct role in regulation of organelle movement during apoptosis. Our studies reveal that TIG3 presence alters the normal subcellular location of microtubules and microfilaments, which may be one mechanism whereby it alters organelle location. Future studies will need to address whether TIG3 also alters microtubule motor-dependent transport of organelles. Our working hypothesis is that TIG3 may alter both microtubule distribution and microtubule-based motor function to cause pericentrosomal organelle clustering. Based on TIG3 Tumor Suppressor our analysis, TIG3 promotes accumulation of organelles at the centrosome including endoplasmic reticulum, Golgi apparatus, recycling endosomes, late endosome, and transport vesicles. However, not all organelles are transported to the centrosome. For example, lysosomes, as measured by detection of LAMP1, distribute in arrays along microtubules and, in TIG3-positive cells, this pattern is intensified (not shown). Thus, additional studies will be necessary to understand the role of TIG3 in regulating microtubule function and organelle transport. Materials and Methods Cell Culture and adenovirus infection SCC-13 cells were obtained from American Type Culture Collection (Rockville, MD) and were maintained in high glucose DMEM (Gibco, 11960-044) supplemented with 2 mM L-gluta- mine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 mg/ml streptomycin and 5% fetal bovine serum (Sigma, St. Louis, MO). Adenoviruses were produced as previously described [10]. tAd5- TIG31–164, is a tetracycline-inducible virus that encodes the full- length TIG3 protein and a tetracycline-responsive enhancer element [10,11]. The Ad5-TA virus encodes the tetracycline transactivator (TA). tAd5-EV is an empty virus used as a control. For infection, cells were washed with PBS, incubated with 10 MOI of TIG3-encoding or empty virus in the presence of 5 MOI of Ad5-TA in DMEM containing 6 mg polybrene/ml (Sigma, H9268). After 5 h, the medium was replaced with virus-free medium and incubation was continued for an additional 24–72 h prior to preparation of cells and extracts for immunohistology and immunoblot. Immunological methods For immunoblot, extracts were prepared in cell lysis buffer (20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 1 mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate, 1 mM sodium vanadate, 1 mg/ml leupeptin (Cell Signaling, 9803, Danvers, MA) and 1 mM phenylmethylsul- fonyl fluoride. Protein concentration was determined using the Bradford Bio-Rad protein assay (Bio-Rad, 500-0006). Equal amounts of protein were electrophoresed on Ready Gels (Bio- Rad, Hercules, CA) and transferred to nitrocellulose membranes for detection of proteins using the appropriate antibodies. For immunofluorescence, cells maintained on coverslips, were infected with adenoviruses and after 24–72 h fixed in 4% paraformalde- hyde, permeabilized with methanol, and incubated with rabbit anti- TIG3 (1:100) and selected organelle marker antibodies followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1000) and Alexa Fluor 555-conjugated goat anti-mouse IgG (1:1000) secondary antibodies. Cells were then incubated with Hoechst 33258 (1:2000), washed thoroughly and mounted on slides using Fluoromount (Sigma, F4680). Confocal cell images were taken using an Olympus IX81 spinning disk confocal microscope. Cell fractionation SCC-13 cells were infected with 10 MOI of tAd5-EV or tAd5- TIG3 and after 24 h the cells were collected and total extract was prepared in cell lysis buffer. A portion of the total extract was set aside for electrophoresis. The remaining extract was centrifuged at 14,0006g and the supernatant and pellet fraction were collected. The pellet fraction was washed once with phosphate-buffered saline and resuspended in gel loading buffer. Total, soluble and pellet fractions were characterized by electrophoresis. PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e23230