Fig 2: Apoptosis induces aS aggregation and phosphorylation in BE(2)-M17D/3D5 cells. a The basic paradigm for cell differentiation, aS induction and staurosporine (STS) treatment. BE(2)-M17D/3D5 cells with 5 days of differentiation and aS induction were exposed to 25 nM STS for 0, 8, 16 and 24 h before harvest. b STS treatment of BE(2)-M17D/3D5 cells induces apoptosis, aS aggregation and Serine 129 phosphorylation, and degradation of lamin B1 (LMNB1) and nuclear pore membrane protein 121 KDa (POM121) in a time-dependent manner. c High dose of STS treatment can rapidly trigger apoptosis and other aforementioned effects which can be significantly prevented by caspase inhibitor. BE(2)-M17D/3D5 cells with 5 days of differentiation and aS induction were exposed to 50 and 100 nM for 4 h before harvest. A group of 100 nM STS-treated sibling cultures was pretreated with caspase inhibitor (CI). All cell lysates were subjected to SDS-PAGE followed by western blotting with antibodies to interesting proteins. Dot blotting of cell lysates was used to detect total aS level (T-aS) for quantification of p-aS/T-aS immunoreactivities. Molecular weight standards were included as references. Arrow denotes a specific cleaved caspase 3 band in BE(2)-M17D/3D5 cells pretreated with caspase inhibitor. Number sign (#) denotes a non-specific Syn1-immunoreactive band on western blots of lysates from BE(2)-M17D/3D5 cells. d, e Statistical analysis of immunoreactivity of various proteins shown in (b and c). Bar graphs in d and e summarize the results of quantitative analyses of immunoreactivities of various proteins in cell lysates from three independent experiments represented by b and c with normalization against ß-actin or T-aS immunoreactivities. The average values of control group (Con or 0 nM) were set as 100 %. Error bars represent standard error of the mean (*p < 0.05, **p < 0.01, comparing to control; #p < 0.05, ##p < 0.01, comparing subsets linked by line, n = 3)

Fig 3: The NLS domain of sPom121 is required for colocalization with Nup98. (A) Schematic of Pom121 mutants used to identify the domain required to localize sPom121 to Nup98 foci. All mutants were cloned from a rat Pom121-3GFP construct that was described previously (Doucet et al. 2010; Talamas and Hetzer 2011). The Pom121 TM domain (blue), NLS domain (red), and FG domain (black) are shown. The fragment of sPom121 required for colocalization with Nup98 is indicated with a black bar (top), while the region that has a minor effect on sPom121 localization is indicated with a dotted black bar (top) (B) IF assays showing localization of the sPom121 mutants listed at the left (panels 1,4,7,10,13,16) and Nup98 (panels 2,5,8,11,14,17). Merged images are shown at the right (in panels 3,6,9,12,15,18). The percentage of cells with the Pom121 mutant colocalizing with GFP-Nup98 in the nucleoplasm is shown in the bottom right corner of the left panels, while the number of cells counted is shown in parentheses. (Panels 3,6,9,12,15,18) The fluorescence intensity of sPom121 and Nup98 at either the nuclear membrane or nucleoplasmic foci was observed by quantifying the intensity of the yellow lines drawn through a cell cross-section. The intensity graphs are shown at the right. (F) Focus. sPom121 only colocalizes with Nup98 in nucleoplasmic foci but never at the NE.

Fig 4: POM121 mRNA expression in 31 OSCC tissue pairs. POM121 mRNA expression was examined by qRT-PCR and normalized to GAPDH. POM121 mRNA levels were higher in the 31 OSCC tissues (T) than that in matched normal tissues (N) (P< 0.05).

Fig 5: Survival curves of OSCC using the Kaplan-Meier method and the log-rank test. (A) Overall survival curves of POM121 (blue line low, green line high).(B) Overall survival curves of TNM stage, TNM 0, I and II (blue line), TNM III and IV (green line). (C) Overall survival curves of location, tongue (blue line), gingival and buccal mucosa (green line). (D) Overall survival curves of tumor size, between 0 and 2 cm (blue line), between 2 and 4 cm (green line), greater than 4 cm (gray line). (E) Overall survival curves of lymph node metastases, N0 (blue line), N1 (green line). (F) Overall survival curves of distant metastases, M0 (blue line), M1 (green line).