Figure 3. Optical sections reveal NPC populations on contra- lateral flattened faces and rim of the nucleus. HEK293 and TOV112D cells were immunolabeled with NUP62 (green) and NUP214 (red) antibodies. Optical sections were generated from the culture plate surface upward to the medium at intervals of 250 nm. The first focused nuclear surface plane (0 um), a central plane, and last focused surface plane were selected and 2D deconvolved. Overlay of NUP62 and NUP214 signals is shown, with coincident signals revealed in yellow. Blue, DAPI stain of nuclei. Bar represents 5 um. doi:10.1371/journal.pone.0036137.g003 In both cell lines, the distributions of NUP62 and NUP214 were consistent between subconfluent and confluent cells (Fig. 5B–D). Spatial distribution of architecturally distinct NPCs varies with cell type Immunoblot analyses (Fig. 6) revealed varied total protein accumulation of both NUP62 and NUP214 in the following cultured cell lines: Hs 832(C).T, COS7, TOV112D, HEK293, and S12. Immunoblot analysis were performed with commercially Diversity in Nuclear Pore Complexes available antibodies (see Materials and Methods) directed at distinct segments of residues on NUP62 (Ab1, human residues 24– 178; Ab2, human residues 401–522) and two distinct segments of residues on NUP214 (Ab3, human residues 2050–2090; Ab4, human residues 1250–1300). For immunofluorescence analyses Ab1 and Ab4 were used as Ab2 and Ab3 did not produce discrete nuclear signals. The absence of a strong immunofluorescence signal for NUP62 Ab2 may result from steric interference, as the epitope(s) for this antibody lie within the coiled-coil domain of NUP62 that directly interacts with other nucleoporins [36]. The epitopes for NUP214 Ab4 and Ab3 lie within the N-terminal border and C-terminal regions of the FG-repeat containing domain, respectively [45]. Ab3 may not produce a signal in immunofluorescence analyses because the binding epitope(s) are obscured by a direct intermolecular interaction that occurs between the C-terminal extension (CTE) and b-propeller struc- tures of NUP214 [45]. The immunoblot signals generated by NUP62 Ab1 and Ab2 were indistinguishable (Fig. 6). Although the immunoblot signals generated by NUP214 Ab3 and Ab4 were similar, difference in the intensity of NUP214 signals detected in Hs 832(C).T and COS7 cells suggests that post-translation modification may reduce binding of Ab4 in Western blots of lysates from these cell lines. The relative abundance and the distribution of NUP62+/ NUP2142, NUP622/NUP214+, and NUP62+/NUP214+ immu- nolabeled NPCs varied between cultured cell types. Ovarian carcinoma TOV112D cells and COS7 cells displayed two major populations of NPCs: peripheral rim NUP62+/NUP2142 and contralateral surface NUP622/NUP214+ NPCs, with few NUP62+/NUP214+ NPCs observed (Fig. 6). In HEK293 cells, NUP62+/NUP214+ NPCs predominated on the rim and contra- lateral surfaces, with some NUP622/NUP214+ NPCs dispersed across the latter (Fig. 6). A human neuroblastoma cell line (S12) displayed NUP62+/NUP2142, NUP622/NUP214+, and NUP62+/NUP214+ NPCs, the latter appearing mostly in segments near the periphery of the nucleus (Fig. 6). The Hs 832(C).T cells, which appear fibroblastoid, displayed few NUP62+/NUP214+ NPCs, with NUP62+/NUP2142 NPCs predominating and fewer NUP622/NUP214+ NPCs on the contralateral nuclear surfaces (Fig. 6). Image stack projections also were viewed from the x,z and z,y planes, and differences in the distribution of NPC populations observed from these perspectives were consistent with those described above. In TOV112D cells, NUP62+/NUP2142 NPCs form an intense belt around the peripheral rim of the nucleus (Fig. 6; x,z and z,y planes). The NUP62+/NUP2142 NPC belt also is apparent at the nuclear rim of COS7 cells, and in HEK293 cells it appears to be populated by NUP62+/NUP214+ NPCs (Fig. 6; x,z and z,y planes). In neuroblastoma (S12) and benign human fibroblastoid (Hs 832(C).T) cells, NUP62+/NUP2142 NPCs appear from two regions of intense immunolabeling on the contralateral flattened surfaces of the nucleus (Fig. 6; x,z and z,y planes). As will be shown below, the intense belts of NUP62 immunolabeling on the nuclear rim observed in TOV112D and COS7 are not evident in S12 and Hs 832(C).T cells, replaced instead by relatively uniform NUP62 immunolabeling of NPCs over the surface of the nucleus. As HEK293 cells displayed a large fraction of NPCs immunolabeled with both NUP62 and NUP214 antibodies (NUP62+/NUP214+ immunolabeled NPCs), binding of both NUP62 and NUP214 antibodies to the same NPC is not precluded if both nucleoporins are present. This supports the interpretation that differences in immunolabeling represent differences in the distribution of NUP62 and NUP214 among NPCs rather than variations in the accessibility of the epitopes. The observed NPC patterns of the cultured cell types were consistent among most of PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e36137