JCS03207 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. (A1) Low-magnification image of asynchronous HeLaS3 cells stained with nuclear pores: a middle section. (A2) A higher magnification image shows bright punctate rim staining on the nuclear equator. (B) Low-magnification image of HeLaS3 nuclear surfaces co-stained with nuclear pores and emerin. Emerin is highly enriched in the pore-free islands with various sizes. Pore-free islands and emerin are localized in an apparent complementary pattern, independent of the sizes of pore-free islands. Note that nuclei even with almost uniform pore distribution show, in details, such complementary pattern (arrowheads). The inset image shows a middle section of emerin staining, demonstrating that emerin forms a typical homogenous ring around the nuclei. (C) Ectopic expressions of CFP-emerin and Venus (YFP-derivative)-P62 in HeLaS3 cells. EF1α-driven CFP-emerin and Venus-P62 were stably expressed in HeLaS3 cells. Although their distributions are not so clear as their signals in immunostaining (Fig. 3 and Fig. S1B) mainly because of their cytoplasmic accumulations, pore-free islands and emerin enrichment are observed.

• Supplemental Figure 2 -

  Fig. S2. (A) Localization of LBR in the pore region. LBR was immunostained by an antibody, demonstrating a clear complementary pattern to the emerin staining. (B) Dynamic pore distributions in human osteosarcoma U-2 OS cells. G1 nuclei (top row) and G2 nuclei (bottom row) co-stained with nuclear pores (Left column) and lamin A/C (center column). The nuclear pores are distributed in a ‘patched’ pattern in early cell-cycle stages, and then become uniformly distributed with cell-cycle progression. Note that lamin A/C is enriched in the pore-free islands (arrows). Merged images of nuclear pores and lamin A/C are shown (right column).

• Supplemental Figure 3 -

  Fig. S3. Left panels: Nuclear pore distribution on the nuclear surfaces in the early G1 stage of human normal diploid fibroblasts IMR90 cells. Condensed chromatins by DAPI staining (Top) and striking ‘patched’ pattern of the pore distribution (bottom) are clearly shown on the nuclear surfaces of the paired nuclei. Right panels: Images of middle section of telophase nuclei co-stained with nuclear pores and lamin A/C. Note that nuclear pores and lamin A/C are recruited to the surface of chromosome clusters (or mass), often in a mutually exclusive manner (shown by arrowheads). 1, DAPI staining; 2, nuclear pores; 3, lamin A/C; 4, merged image of 2 and 3.

• Supplemental Figure 4 -

  Fig. S4. (A) Enlarged images of early G1 nuclei of HeLaS3 cells co-stained with Ki-67 and nuclear pores. Ki-67 punctate foci are observed in the early G1 nuclei (panel 3). Note that many Ki-67 foci are often accompanied by bright foci of DAPI staining (shown by arrowheads in panels 2-4). 1, nuclear pores; 2, DAPI; 3, Ki-67; 4, merged image of 2 and 3; 5, merged image of 1 and 3. (B) Enlarged images of mid-late S-phase nuclei co-stained with PCNA and nuclear pores. In the mid-late S phase, large PCNA foci are restricted to the nuclear periphery (panels 2 and 5). Note that such PCNA foci are often located in the small pore-free areas (3 and 6). Left (1 and 4), nuclear pores; center (2 and 5), PCNA; right (3 and 6), merged images.

• Supplemental Figure 5 -

  Fig. S5. (A) Localization of ER in the HeLa S3 cells. HeLaS3 cells were immunostained by an anti-calnexin antibody, demonstrating that nuclei are closely surrounded with ER. The pore-free islands are, however, almost excluded from ER. (B) In vivo incorporation of Br-UTP in digitonin-permeabilized HeLaS3 cells. To detect transcriptional activity, in vivo-incorporation of Br-UTP in digitonin-permeabilized HeLaS3 cells was measured. Levels of incorporation in the nuclei with large pore-free islands were significantly lower than the nuclei with uniform pore distribution, suggesting a correlation between nuclear pore density and transcriptional activity. (C) Growth curves of HeLa, HeLaS3 and U-2 OS cells. Note that HeLa cells with lower amounts of lamin A/C and uniform pore distribution (Fig. 4A), have a higher proliferation activity than HeLa S3 and U-2 OS cells.