Knockdown of NUP62 or NUP214 in ovarian carcinoma cells (TOV112D) treated with specific siRNAs.

<p>A. Constant total protein from siRNA-treated cultures was analyzed for nucleoporin content by immunoblot with the indicated antibodies. Dilutions of the cultures treated with control siRNA are shown for comparison to semi-quantify the extent of knockdown (siRNA knockdown resulted in approximately the same signal intensity as dilution to 25%). B. Immunofluorescence microscopy of TOV112D cells treated with NUP62 or NUP214 siRNAs. Immunofluorescent images represent 3D deconvolved projections of 10–15 um total of optical sections through the z axis. C. Immunofluorescence microscopy of NUP133 and NUP62 or NUP214 in TOV112D cells. Bars represent 2 um.</p

Detection of NUP62 and NUP214 in nuclear and cytoplasmic compartments of cultured cells.

<p>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.</p

Effects of transfection reagents and cell density on nucleoporin distribution.

<p>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.</p

Optical sections reveal NPC populations on contralateral flattened faces and rim of the nucleus.

<p>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.</p

Radial intensity distribution graphs of NUP62 and NUP214 immunofluorescence in cultured cell lines.

<p>Immunofluorescent images for fields of TOV112D, HEK293 (293), COS7, or S12 cells (similar to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036137#pone-0036137-g005" target="_blank">Fig. 5</a>), and images from several different Hs 832(C).T cells, were analyzed. Radial intensity distributions for NUP62 or NUP214 were determined for each cell line and normalized to arc length, and a minimum of 24 sectors derived from at least six independent nuclei were used for each analysis. Radial distances were normalized from 0 (point of origin) to 1 (surface of nuclear envelope). Distribution points are colored according to residuals (blue: less than one standard error; green: less than two standard errors; red: greater than two standard errors). Plotted in red are 95% confidence intervals.</p

Immunolabeling patterns of native NUP62 and NUP214 antibodies are consistent with those produced by V5 epitope tag antibodies detecting ectopically expressed nucleoporins.

<p>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 10 um. A. TOV112D cells were transfected with an expression plasmid for V5 epitope-tagged NUP62 and cultured for 72 hours. The distribution of NUP62 was analyzed in pairs of post-mitotic cells expressing V5 epitope-tagged NUP62. Immunofluorescence analyses of NUP62 (green) and V5 epitope-tagged NUP62 (red) were performed with Ab1 and V5 epitope tag antibodies, respectively. Blue, DAPI stain; yellow, overlap between green and red. B. TOV112D cells were transfected with an expression plasmid for V5 epitope-tagged NUP214 and cultured for 72 hours. The distribution of NUP214 was analyzed in pairs of post-mitotic cells expressing V5 epitope-tagged NUP214. The images of the pair of post-mitotic cells shown have been cropped individually to avoid interference from surrounding cells, particularly on the x,z and z,y planes. Immunofluorescence analyses of NUP214 (red) and V5 epitope-tagged NUP214 (green) were performed with Ab4 and V5 epitope tag antibodies, respectively. Blue, DAPI stain; yellow, overlap between green and red. C. TOV112D cells were transfected with an expression plasmid for V5 epitope-tagged NUP214 and cultured for 72 hours. The distributions of NUP62 and NUP214 were analyzed in pairs of post-mitotic cells expressing V5 epitope-tagged NUP214. Immunofluorescence analyses of NUP62 (green) and V5 epitope-tagged NUP214 (red) were performed with Ab1 and V5 epitope tag antibodies, respectively. Blue, DAPI stain; yellow, overlap between green and red.</p

Total accumulation and distribution of NUP62 and NUP214 in different cell lines.

<p>Top left corner: immunoblot analyses of Hs 832(C).T (Hs), TOV112D (112D), HEK293 (293), S12, and COS7 (cos7) cell lysates. Antibodies are explained in the text. Immunofluorescent images for the indicated cell lines 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. immunofluorescence analyses of NUP62 (green) and NUP214 (red) were generated using Ab1 and Ab4, respectively. Blue, DAPI stain; yellow, overlap between green and red. Bars represent 2 um.</p

Optical sections of NUP62 and NUP214 immunolabeling in S12 cell nuclei using confocal and high resolution STED microscopy.

<p>HIgh resolution STED (NUP214; red) and standard confocal (NUP62; green) double immunofluorescence microscopy were performed on S12 neuroblastoma cells. Individual optical sections were selected from the ventral (culture plate side) nuclear surface (0 um) upward to the dorsal surface at intervals of 250 nm. Yellow, overlap bewteen green and red. Bar represents 5 um.</p

High resolution STED microscopy resolves NPC clusters, reveals microheterogeneity in NPC populations, and demonstrates that NPCs from different populations are similar in size.

<p>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, 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.</p

Nuclear distribution of NPC populations varies with cell type.

<p>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.</p