TIG3 Tumor Suppressor Figure 6. Pericentrosomal organelle accumulation in TIG3-positive cells. A SCC-13 cells were infected by 10 MOI tAd5-EV or tAd5-TIG3 and after 24 h fixed and stained to detect TIG3 (green) and various organelle markers (red). The markers include GM130 (cis-Golgi), M6PR (mannose-6- phosphate receptor, trans-Golgi and late endosome), rab11 (recycling endosome), calnexin (ER), and CHC (clathrin heavy chain, intracellular transport vesicle). White arrows indicate pericentrosomal localization of TIG3. Nuclei are stained blue. For GM130, M6PR, and rab11, all panels are red/green/ blue merged images. The EV and TIG3 pictures of calnexin staining are red/green/blue merged images. The inserts in the TIG3/calnexin picture are single-color images. For the CHC stain, only the red (CHC) image is shown. Bars = 10 mm. B TIG3 impact on subcellular organelle distribution. To measure organelle spread, the microscope was focused on the z-plane displaying the maximal organelle spread and the area covered by the organelle was monitored using ImageJ Software and presented as organelle area in mm2. The values are mean 6 SEM (n = 30 fields) including measurement of a minimum of thirty cells per treatment group. Paired Student’s t-test analysis identifies the means as significantly different (p,0.001). doi:10.1371/journal.pone.0023230.g006 For example, the TIG3-dependent increase in p21 protein is as- sociated with a parallel increase in p21 encoding mRNA, indicating that TIG3 regulates p21 gene transcription or RNA stability. We do not presently know whether this action is direct or indirect. TIG3 expression is associated with pericentrosomal organelle clustering An important question is where TIG3 is localized in the cell. An interesting previous study in HeLa cervical cancer cells suggests that TIG3 localizes in the cis- and trans-Golgi and that this localization is required to stimulate apoptosis [2]. As shown in Fig. 6A, antibody co-staining of SCC-13 cells suggests that TIG3 localizes in the cis- and trans-Golgi (GM130, M6RP), the late endosome (M6PR), the recycling endosome (rab11), the endoplas- mic reticulum (calnexin) and the intracellular transport vesicles (CHC). However, we believe that is it unlikely that TIG3 is principally localized in these structures for several reasons. First, TIG3 only appears to localize with the organelle only in the immediate vicinity of the centrosome and not more peripherally. Second, we show that TIG3 alters microtubule and microfilament distribution and this is associated with pericentrosomal organelle accumulation. Third, in another report we are studying the PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e23230