Fig 2: Effects of transfection reagents and cell density on nucleoporin distribution.A. TOV112D cells were treated in the absence of exogenous DNA with Lipo293D (SignaGen Laboratories), Lipofectamine (Invitrogen), or FuGENE HD (Promega), according to the instructions provided by the manufacturers. The cells were processed for immunofluorescence using NUP62 (green) and NUP214 (red) antibodies 48 hours after treatment. Immunofluorescent images represent 3D deconvolved projections of 250 nm optical sections through 10 to 15 um of the z axis. Projections are shown on x,y, x,z, and z,y planes, as indicated. Bar represents 5 um. Blue, DAPI stain; yellow, overlap between green and red. B. TOV112D were cultured at confluence for three days, and analyzed by immunofluorescence microscopy as described above. C. TOV21G cells were cultured at confluence for three days, and analyzed by immunofluorescence microscopy as described above. D. TOV21G cell cultured at low density and analyzed by immunofluorescence microscopy as described above.

Fig 3: Nuclear distribution of NPC populations varies with cell type.Double immunofluorescence analyses of NUP62 (green) and NUP214 (red) are shown for large fields of subconfluent TOV112D ovarian carcinoma, COS7, S12 neuroblastoma, and HEK293 cells. Blue, DAPI stain; yellow, overlap between green and red. Bar represent 10 um.

Fig 4: 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.

Fig 5: High resolution STED microscopy resolves NPC clusters, reveals microheterogeneity in NPC populations, and demonstrates that NPCs from different populations are similar in size.HIgh resolution STED microscopy (STED) and confocal (Conf.) double immunofluorescence microscopy were performed with NUP62 (green) and NUP214 (red) antibodies in S12 neuroblastoma cells. All images are projections of 8–10 um total optical z-stacks. Bars represent 500 nm. A. Comparison of confocal and STED microscopy resolution of NUP214 antibody immunofluorescence. A and B. Arrows, NUP62+/NUP214− NPCs; asterisks, NUP62>NUP214 NPCs; diamonds, NUP62+/NUP214+ NPCs; triangles, NUP62arrowheads, NUP62−/NUP214+ NPCs. C, D, and E. Measurement of diameters of NUP62+/NUP214− (C), NUP62−/NUP214+ (D), and NUP62+/NUP214+ (E) NPCs in S12 cells. Measured pores were derived from STED microscopy images (indicated by asterisks). Yellow represents overlap between green and red.