Figure 2. TIG3 reduces cell cycle progression. A SCC-13 cells grown on coverslips were infected with 10 MOI of EV or TIG3-encoding virus and after 24 h treated with 10 mM BrdU for 2 h and then fixed and stained with anti-BrdU (red) and anti-TIG3 (green). BrdU incorporation is a marker of the synthesis phase of the cell cycle. The number of BrdU positive cells as a percentage of total cell number is presented beneath each panel. The values are mean 6 SEM (n = 3) and the values are significantly different as determined by Student’s t-test (p,0.001). Bars = 10 mm. B SCC-13 cells were collected for flow cytometry at 24 h post-infection with EV or TIG3-encoding virus. Cells were stained with 50 mg/ml propidium iodide prior to analysis. TIG3 reduces events in G1 and increases subG1 events. C At 24 h post-infection, cells were harvested and extracts prepared for detection of cell cycle regulatory proteins. Molecular weights are indicated to the left of the blot in kDa. D Cells were treated as above and then harvested for detection of p21 encoding mRNA by real time-PCR. A similar result was observed in each of three experiments. doi:10.1371/journal.pone.0023230.g002 radiate out from the centrosome, suggesting that TIG3 impacts microtubule location and anchorage. This is best observed in Fig. 5A (lower panel) which shows only the b-tubulin staining. Microtubules in the TIG3-positive cell (left) lack centrosome-based clustering, while the TIG3-negative cell (right) has centrosome- anchored microtubules (white arrow). To assess the impact of TIG3 Tumor Suppressor TIG3 on the microtubule network, the number of cells displaying microtubules radiating from the centrosome was counted. As shown in the plot, the percentage of cells displaying centrosome- directed networks is markedly reduced in TIG3-expressing cells. The redistribution of tubulin to the cell membrane in TIG3- positive cells suggests that it may be insoluble. To assess this, we prepared total cell extract from TIG3-negative and positive cells and prepared soluble and particular (pellet) fractions. We then assayed for b-tubulin in each fraction by immunoblot. As shown in Fig. 5A, the membrane-localized b-tubulin does not appear in the pellet fraction, suggesting that although it is localized at the cell periphery at or near the membrane, it remains soluble. The tubulin and actin networks interact extensively and so we monitored the impact of TIG3 on actin filament distribution (Fig. 5B). In TIG3-negative cells (EV), b-actin (red) distributes throughout the cytoplasm. In contrast, in TIG3-positive cells (green stain) a distinct actin filament ring forms at the nuclear periphery (red arrow). This is shown in both a merged image and b-actin staining alone (Fig. 5B). The number of cells displaying a nuclear actin ring was counted. The plot shows that the percentage of cells displaying a nuclear actin ring is markedly increased in TIG3-expressing cells. Because organelle movement and anchoring depend upon the tubulin and actin networks [29], we anticipated that TIG3 may influence organelle distribution. To assess the impact of TIG3 on organelle location, cells were stained to detect GM130 (cis-Golgi), mannose-6-phosphate receptor (M6PR, trans-Golgi and late endosome) [30], rab11 (recycling endosomes), calnexin (endoplas- mic reticulum), and clathrin heavy chain (CHC, intracellular transport vesicles). Fig. 6A shows that GM130 (red) localizes at a perinuclear location in TIG3-negative and positive cells; however, it appears more compacted in TIG3-positive (green stain) cells. M6PR, rab11 and calnexin also appear compacted at the pericentrosomal location in TIG3-positive cells (white arrows). This is readily visualized for calnexin, by comparing the dis- tribution shown in the red single-color image (insert) with that observed in EV-infected cells (Fig. 6A), as an intense pericen- trosomal concentration of calnexin is observed in TIG3-positive cells. In addition, clathrin heavy chain is a particularly dramatic example, in that staining appears distributed throughout the cytoplasm in EV-infected cells, but essentially all of the CHC accumulates in the vicinity of the centrosome in TIG3-positive cells. Thus it appears that TIG3 alters intracellular organelle distribution in a manner that concentrates and compresses many organelles in the vicinity of the centrosome. This compaction was quantified by measuring the two-dimensional area covered by individual organelles in square microns using ImageJ Software (Fig. 6B). This analysis reveals a substantial and statistically significant reduction in area for GM130, M6PR and rab11. Calnexin and CHC distribution was not analyzed in this manner, since the condensation was obvious. We also examined the impact of TIG3 on organelle protein level. Fig. 7 shows that TIG3 expression causes a modest reduction in the level of some organelle proteins, including calnexin, rab11, GM130 and EEA1; however, the level of other marker proteins is not altered. Discussion TIG3 (Tazarotene-induced gene 3) was originally identified as increased following treatment of cultured epidermal keratinocytes or psoriatic epidermis with the synthetic retinoid, Tazarotene [6], and was later identified in gastric cancer cells and called RIG1 [5]. Subsequent studies reveal TIG3 mRNA in a range of tissues and cells in culture [1,5,31–33]. TIG3 level is increased by retinoid PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23230