TIG3 Tumor Suppressor Figure 3. TIG3 induces apoptosis. A SCC-13 cells were infected with 10 MOI of EV or TIG3-encoding virus and after 24 h lysates were prepared for detection of apoptosis markers. The bracket indicates high molecular weight crosslinked TIG3 [10,11]. Molecular weights are indicated to the left of the blot in kDa. B At 24 h post-infection SCC-13 cells were fixed and stained with anti-cleaved PARP (red). TIG3 increases cleaved PARP staining. The percentage of cleaved PARP positive cells is presented in each panel (mean 6 SD). The arrows indicate cleaved PARP-positive cells. Similar results were observed in each of three experiments. doi:10.1371/journal.pone.0023230.g003 Figure 4. TIG3 localizes in the vicinity of pericentrin. A SCC-13 cells were infected with 10 MOI tAd5-EV or tAd5-TIG3 and at 24 h post- infection were fixed and stained with anti-TIG3 (green). Arrows indicate pericentrosomal and arrowheads indicate membrane localization. No TIG3 is detected in empty vector-infected cells. B Cells, infected as above, were fixed and stained with TIG3 (green) and pericentrin (red). The arrows indicate pericentrin staining of the centrosome and n indicates the nuclei. Bars = 10 mm. doi:10.1371/journal.pone.0023230.g004 treatment [4,34] and in some cell types TIG3 gene expression is suppressed by MAPK signaling [35]. TIG3 is a member of the NIpC/P60 superfamily of proteins [36]. The N-terminal domain (aa1–134) encodes regions that are conserved among members of the lecithin:retinol acyltransferase (LRAT) and H-rev tumor suppressor families [37–39]. Function- al analysis of the TIG3 structure indicates that the c-terminal hydrophobic domain (aa135–164) functions as a membrane anchor [6,23] and that the N-terminal region encodes calcium-independent phospholipase A1/2 activity [31,32,40]. Moreover, phospholipase A1/2 activity does not require the c-terminal hydrophobic domain [40]. TIG3 has been shown to reduce cell survival in a number of cell types [6,10,11,34,41,42], but the mechanism responsible for the suppression is not well understood. In some cell types, TIG3 may act via regulation of MAPK and PI3K/Akt signaling [1,3,41]. TIG3 in epidermis TIG3 is expressed in the suprabasal differentiated layers of keratinocyte raft cultures [12] and in suprabasal epidermis [7,8], and TIG3 expression is reduced in hyperproliferative skin disease and in skin cancer cells [6–9]. Retinoid treatment increases TIG3 level and this is associated with reduced cell proliferation [6–9]. TIG3 is not expressed in normal keratinocytes maintained in monolayer culture and vector-mediated TIG3 expression is associated with reduced cell number [8,10–12]. Mechanistic studies in keratinocytes show that the c-terminal membrane anchoring domain is required for activity [10,11,23], and that expression of TIG3 in normal human cultured keratinocytes activates selected differentiation-related events [10–12]. In particular, TIG3 interacts with type I transglutaminase (TG1) to increase TG1 catalytic activity [10,11]. TG1 is a key enzyme in differentiating keratino- cytes that catalyzes formation of protein-protein crosslinks leading to assembly of the cornified envelope, an important component of the skin permeability barrier [43]. Full-length TIG3 protein stimulates differentiation-associated transglutaminase activity in keratinocytes. In contrast, amino-terminal truncated forms cause PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23230