TIG3 Tumor Suppressor Figure 5. TIG3 alters microtubule distribution. A SCC-13 cells were infected with 10 MOI tAd5-EV or tAd5-TIG3 and at 24 h post-infection cells were fixed and stained with anti-TIG3 (green stain) and anti-b-tubulin (red stain). TIG3 accumulates at the expected perinuclear location (black arrow). b-tubulin accumulates in an atypical ring at the cell periphery (red arrows). The normal b-tubulin network in the TIG3-negative cell is indicated by a white arrow pointing to the centrosome. Nuclei were Hoechst stained (blue). The bottom panel is identical to the top, except that only the b-tubulin (red) signal is indicated. Bars = 10 mM. The graph shows the number of tAd-EV and tAd5-TIG3 infected cells with centrosome-originated microtubule arrays. Cells were counted in twenty independent microscope fields and a minimum of one-hundred cells were counted per treatment group. The values are mean 6 SEM. Paired Student’s t-test analysis reveals that the means are significantly different (p,0.001). To assess the impact of TIG3 on tubulin solubility, cells were infected with tAd5-EV or tAd5-TIG3 and after 24 h total extract, soluble and pellet fractions were prepared and electrophoresed followed by immunostaining to detect b-tubulin and b-actin. The presence of the majority of b-actin in the soluble fraction indicates that the fractionation was successful. B TIG3 expression causes actin filament collapse around the nucleus. SCC-13 cells were infected with adenovirus as above and after 24 h stained with anti-b-actin (red stain) and anti-TIG3 (green stain). Nuclei were Hoechst stained (blue). For the TIG3-positive cells, the left panel shows the TIG3 and b-actin signals (red and green), while the right panel shows only the b-actin (red) channel. The black arrow indicates TIG3 accumulation at the centrosome and the red arrow indicates the b-actin microfilament nuclear ring. Bars = 10 mm. The plot shows the number of tAd-EV and tAd5-TIG3 infected cells displaying actin microfilament rings surrounding the nucleus. Cells were counted in eighteen independent microscope fields and a minimum of one-hundred cells were counted per treatment group. The values are mean 6 SEM. Paired Student’s t-test analysis reveals that the means are significantly different (p,0.001). doi:10.1371/journal.pone.0023230.g005 apoptosis and the level of apoptosis is more pronounced as the N- terminus is shortened [12]. Other studies point to unique effects of various TIG3 subdomains [40,42,44]. The suprabasal pattern of TIG3 expression in epidermis is consistent with a role of TIG3 as a controller of keratinocyte differentiation-related events. TIG3 expression in skin cancer cells reduces proliferation and increases apoptosis Previous studies indicate that TIG3 mRNA is present at reduced levels in skin cancer and in skin cancer cell lines [8]. However, little is known regarding whether TIG3 regulates skin cancer cell survival and tumor progression. We show that expression of TIG3 causes a marked reduction in SCC-13 cell number that is associated with reduced G1 and S phase events and increased sub-G1 DNA content. These cell cycle changes are associated with TIG3-dependent changes in cell cycle regulatory protein level. TIG3 expression reduces cyclin D1 and cyclin E levels and increases the level of the p21 cyclin-dependent kinase inhibitor. These findings are consistent with a reduction in cell progression through the G1/S transition. In addition, we demonstrate that TIG3 increases SCC-13 cell apoptosis as evidenced by increased production of activated caspase 9 and 3 and increased cleaved PARP. Moreover, immunostaining studies reveal cleaved PARP accumulates in TIG3-positive cells. These results are particularly interesting as TIG3 does not cause apoptosis in normal human keratinocytes. Instead, TIG3 causes the cells to undergo differentiation [10–12]. In contrast, mutant forms of TIG3 cause apoptosis in normal human keratinocytes [12]. The fact that TIG3 causes apoptosis in cancer cells suggests a different mechanism of action in normal versus transformed cells. In addition, some of these changes are associated with changes in target gene mRNA level. PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e23230