Diversity in Nuclear Pore Complexes Figure 2. Detection of NUP62 and NUP214 in nuclear and cytoplasmic compartments of cultured cells. Whole cell, cytosolic, and nuclear fractions from Hs 832(C).T (Hs), TOV112D, HEK293 (293), S12, and COS7 cells were analyzed by immunoblot with antibodies to NUP62 and NUP214 (antibodies used were also used for immunofluorescence). Two T75 plates of cells at 80% confluence were used for each preparation (this does not yield equal numbers of cells). One third of the sample was taken for the whole cell extract; the remaining sample was fractionated into cytosol and nuclear components. Extracts were loaded to represent equivalent fractions of starting material. Indicated as Mr,35K is an additional band observed in the NUP214 immunoblot. The Mr,70K band present in the Hs832(C).(Hs) whole cell lysate fractionated with the cytosol. The Western blot analyses of the whole cell lysates were completed on single membranes for all five cell lines; however, the exposure times for Hs832(C).(Hs) and Cos 7 cells using the NUP214 antibody and for Hs832(C).(Hs) using the NUP62 antibody were longer. Consequently, those lanes are separated. doi:10.1371/journal.pone.0036137.g002 in the central optical section from TOV112D cell nuclei produced the typical punctate nuclear rim pattern with NUP62 antibody, whereas NUP214 antibody produced dense accumulations of intranuclear labeling and a comparatively weak signal from the nuclear rim (Fig. 3). The NUP214 intranuclear pattern clearly does not represent NPCs as it is neither punctate nor resident on the nuclear surface. Punctate NUP62 and NUP214 immunolabel- ing observed on the contralateral surfaces of TOV112D cell nuclei displayed only partial overlap. Further studies of NUP62 and NUP214 immunolabeling in TOV112D cells were performed using expression constructs for V5 epitope-tagged NUP62 or NUP214 cDNAs. Plasmids were transfected into TOV112D cells, and processed for immunoflu- orescence microscopy 72 hours later using NUP62, NUP214, and/or V5 epitope tag antibodies. Observations were restricted to pairs of post-mitotic cells. As NPCs and the nuclear envelope are disassembled during mitosis then reassembled during abscission and early G1 phase, observation of post-mitotic pairs of transfected cells assures that the V5 epitope-tagged forms of NUP62 and NUP214 have distributed into the total NPC and nucleoporin pools. Cells transfected with V5 epitope-tagged NUP62 or NUP214 expression constructs were analyzed by immunofluorescence with V5 epitope antibody and NUP62 or NUP214 antibodies, respectively. Extensive co-localization of NUP62 or NUP214 immunolabeling with V5 epitope tag immunolabeling was observed in transfected TOV112D cells (Fig. 4A, B), confirming signal specificity of the nucleoporin antibodies. In transfected cells, NUP62 and NUP214 antibody immunolabeling was slightly more extensive than that of the V5 epitope antibody, as the nucleoporin antibodies bind both the transfected V5 epitope-conjugated and endogenous forms of NUP62 and NUP214. As expected, total NUP62 or NUP214 immunolabeling increased in cells transfected with expression constructs (Fig. 4A, B). The V5 epitope tags were inserted at the N-termini of NUP62 and NUP214. The NUP62 antibody recognizes an epitope within residues 24–178 and the NUP214 antibody recognizes epitopes between residues 1250–1300 of the human form of the protein. The nucleoporin immunolabeling patterns generated by native and epitope tag antibodies overlapped extensively, suggesting that immunofluorescence patterns generated by the native NUP62 or NUP214 antibodies reflect the presence or absence of nucleopor- ins and not post-translational modifications that alter antibody binding (Fig. 4A, B). The TOV112D cells were transfected with V5 epitope-tagged NUP214 expression construct, and analyzed by immunofluorescence microscopy 72 hours later using NUP62 and V5 epitope tag antibodies. The appearance of nuclei immunola- beled in this manner resembled those in untransfected cells immunolabeled with NUP62 and NUP214 antibodies: intense NUP62 signal near the nuclear rim, and weaker V5 epitope- tagged NUP214 signal at the rim accompanied by common and distinct labeling at the contralateral nuclear surfaces (Fig. 4C). This result supports the differential distributions of NUP62 and NUP214 observed using native NUP214 antibody. The enhanced level of V5 epitope-tagged NUP214 signal observed at the nuclear rim of transfected cells is likely to result from the higher level of total NUP214 expression. Changes in the distribution of NUP214 were observed using Lipo293D (SignaGen Laboratories) transfec- tion reagent in the presence or absence of DNA, but were not observed when other transfection reagents were used, including Lipofectamine (Invitrogen). While Lipo293D is a liposome-based reagent, it contains a proprietary booster peptide that targets liposomal contents to the nucleus and greatly increases transfection efficiency. Accumulation of NUP214 in loci much larger than NPCs was observed after Lipo293D treatment; by observing image stacks from x,y, x,z and z,y perspectives, it is clear that these loci are intranuclear (Fig. 5A). It is intriguing to speculate from this observation that the presence or abundance or certain NPC cargo, such as the nuclear localization peptide present in Lipo293D, can directly influence the nuclear distribution of NUP214. We also asked whether the density of cells influences NUP62 and/or NUP214 immunolabeling patterns. Consistent with their origin in a high grade ovarian carcinoma, TOV112D cells form a poor monolayer with abundant overgrowth and cell death, whereas TOV21G cells (a cell line derived from another ovarian carcinoma; [41]) form a monolayer with an epithelial appearance. PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e36137