Fig 1: Nup153 and PRC1 components associate with and co-occupy the TSSs of several differentiation genes. (A) Pie chart showing the percentage of Nup153 DamID-seq peaks co-occupied by Ring1b and/or Cbx7 peaks. (B) Venn diagram showing overlap of Nup153-, Ring1b-, and Cbx7-bound genes in mESCs. Bound genes are defined as having occupancy peaks between −2 kb from the TSS and +1 kb from the TES. (C) Ring1b ChIP-seq read density per TSS at Nup153-bound TSSs (blue) versus unbound TSSs (red). (D) Representative differentiation gene (Nestin) containing a Nup153 DamID-seq peak and a Ring1b ChIP-seq peak at the TSS. For visualization, the biological replicates used to generate the Nup153 and GFP DamID profiles were merged. A schematic illustration of the genomic structure of the gene loci, its genomic location, and the associated GATC content is also shown. (E) Ring1b ChIP-seq, Cbx7 ChIP-seq, and Nup153 DamID-seq profiles at the TSSs of representative developmental genes. (F) Anti-Flag immunoprecipitation of Flag.GFP, Flag.mCherry.Nup153, and Flag.GFP.Nup50 ectopically expressed in mESCs followed by Western blot using antibodies against Nup153, Ring1b, and Cbx7.
Fig 2: SMARCC1 is a cargo protein of KPNA2 in BC cells. (A) Pearson’s correlation analysis indicated a distinct positive correlation between SMARCC1 and KPNA2 expression. (B) A Co-IP assay was used to examine the association between KPNA2 and SMARCC1 in vitro. (C–D) After knockdown of KPNA2, Nup50 and Nup153, SMARCC1 expression was dramatically decreased in the nuclear fraction, while increased in the cytoplasmic fraction of UMUC-3 cell. (E) Representative immunofluorescence (IF) staining images, showing SMARCC1 (green) and KPNA2 (red) expression in the UMUC-3 cell in bladder cancer. Scale bar, 20 μm.
Fig 3: Nup153-mediated silencing of specific developmental genes in mESCs seems to be independent of its roles in global nuclear transport. (A) Immunofluorescence for the pluripotency marker Oct4 in control and Nup153 knockdown mESCs after 6 d of Nup153 knockdown. Bars, 10 μm. (B) Representative image from a Nup153 IF-RNA-FISH for poly(A)+ RNAs in control and Nup153 knockdown mESCs after 6 d of Nup153 depletion. Bars, 10 μm. (C) Western blot analysis for Nup153 and Nup50 in whole-cell extracts from control, Nup153 knockdown, and Nup50 knockdown mESCs upon 5 d of depletion. Gapdh was used as the loading control. (D) Western blot analysis for Nup153 and Nup107 in whole-cell extracts from control, Nup153 knockdown, and Nup107 knockdown mESCs upon 7 d of depletion. Tubulin was used as the loading control. (E) NPCs were stained and visualized by superresolution microscopy, and individual pores were counted for each nucleus. N ≈ 30. Maximum projection of representative reconstructed images (left panel) and total number of single pores per cell (right graph) from control, Nup153 knockdown, and Nup107 knockdown nuclei. P-values were obtained from Student's t-test; (***) P < 0.001. (F) Representative phase-contrast microscopy images of control, Nup153 knockdown, and Nup50 knockdown AP stem cell colonies. Original magnification, 100×. (G) Representative phase-contrast microscopy images of control, Nup153 knockdown, and Nup107 knockdown AP stem cell colonies. Original magnification, 100×. (H) qRT–PCR analysis of four different ectodermal markers in control, Nup153 knockdown, and Nup50 knockdown mESCs after 5 d of knockdown. The relative expression levels were normalized to actin and are expressed as fold change relative to the control (shCTRL). (I) qRT–PCR analysis of four different ectodermal markers in control, Nup153 knockdown, and Nup107 knockdown mESCs after 7 d of knockdown. The relative expression levels were normalized to actin and are expressed as fold change relative to the control (shCTRL). The mean ± SD from three independent experiments is shown. P-values were obtained from Student's t-test; (***) P < 0.001.
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