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242 importin α3  (Novus Biologicals)


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    Novus Biologicals 242 importin α3
    242 Importin α3, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/kpna3/KPNA3+Antibody/pmc11461878__41398_2024_3138_MOESM1_ESM-101-65-70
    Average 92 stars, based on 1 article reviews
    242 importin α3 - by Bioz Stars, 2026-10
    92/100 stars

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    Article Title: mRNA export through an additional cap-binding complex consisting of NCBP1 and NCBP3
    Article Snippet: Streptavidin-agarose beads were obtained from Novagen, GFP-Trap-coupled agarose beads were from Chromotek, and γ-Aminohexyl m 7 GTP- and GTP-agarose beads from Jena Bioscience and Biorbyt, respectively.

    Western Blot:

    Article Title: mRNA export through an additional cap-binding complex consisting of NCBP1 and NCBP3.
    Article Snippet: Streptavidin-agarose beads were obtained from Novagen, GFP-Trap-coupled agarose beads were from Chromotek, and g-Aminohexyl m7GTP- and GTP-agarose beads from Jena Bioscience and Biorbyt, respectively. .. Primary antibodies (1:1000 dilution for western blotting if not stated otherwise) used in this study were as follows: C17orf85 (Atlas Antibodies; HPA008959), NCBP1, SRRT, EIF4A3 and ALYREF (Thermo Scientific; PA 5–30098, PA5–31593, PA5–30329 and MA 1–26754), THOC5, PHAX and KPNA3 (Novus Biologicals; NBP1–19160, NBP2–22268 and NB100–81650), GFP (Invitrogen; A6455), PABP1 and b-tubulin (1:500 dilution for western blotting) (Santa Cruz; 10E10, sc-9104), histone H3 (Abcam; ab1791–100) and EIF4E (Cell Signaling; C46H6). ..



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    <t>KPNA3</t> interacts with the NLS of NPAT. (A) MCF-7 cells treated with or without ivermectin (25 μM) were stained with phalloidin for actin and NPAT antibody for HLB. Bar: 10 μm. The insets show the magnification of the boxed region (scale bar, 5 μm). The numbers of HLB foci per cell were quantified as violin plots (DMSO: n = 126; ivermectin: n = 132). ****: P < 0.0001. (B) MCF-7 cells treated with or without ivermectin (25 μM) were subjected to qPCR analysis for transcription levels of the indicated RD histone genes and two control genes. Data in the bar graphs represent the means ± SEM values of the relative transcription levels for three independent experiments. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant. (C) Confocal images of 293T-GFP-NPAT cells treated with or without ivermectin (25 μM). Bar: 10 μm. The insets show the magnification of the boxed region (scale bar, 5 μm). The GFP-NPAT foci per cell were quantified as violin plots (DMSO: n = 85; ivermectin: n = 73). ****: P < 0.0001. (D) Coimmunoprecipitation of FLAG-NPAT and HA-tagged KPNA family members. (E) Schematic diagram of NPAT and its truncation mutants. (F) Coimmunoprecipitation of FLAG-tagged NPAT truncation mutants and GFP-KPNA3. (G) Schematic diagram of the NPAT C-region and its ΔNLS deletion mutant (Δ1,368–1,405). (H) Confocal images of representative MCF-7 cells expressing the GFP-NPAT C-region and its NLS deletion mutant. Scale bar: 5 μm. Data in the bar graphs represent the means ± SD values of the ratio of nuclear GFP signal of WT or NLS deletion mutant for three independent experiments. ****: P < 0.0001. (I) Coimmunoprecipitation of the FLAG-tagged NPAT C-region or its ΔNLS deletion mutant and HA-KPNA3. Source data are available for this figure: .
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    TMUV-NS5 interacts with KPNAs, resulting in the reduction of pSTAT1 nuclear traffic. ( A ) HeLa and HEK293 cells were co-transfected with the indicated plasmids of Vec (HA), HA-NS5, and Myc-KPNA1. At 24 h post-transfection, cells were treated with 1,000 U/mL IFN-α for 30 min, and then IP was performed with an anti-Myc antibody and subsequently IB with the pSTAT1 antibody. ( B ) HeLa cells were transfected with the HA-NS5 plasmid or Vec (HA). Twenty-four hours after transfection, cells were stimulated with IFN-α (1,000 U/mL) for 30 min, and the expression levels of endogenous KPNA1, KPNA2, KPNA3, KPNA4, KPNA5, and KPNA6 were analyzed by WB. ( C ) HeLa cells were treated with IFN-α (1,000 U/mL) for 30 min in the presence or absence of HA-NS5, and IP was conducted with anti-HA antibodies, subsequently IB with KPNA1, KPNA2, KPNA3, KPNA4, KPNA5, and KPNA6 antibodies. An empty vector of pCAGGS-HA was used as a control. ( D ) HA-NS5 and Myc-KPNA1 (with the empty vector of pCMV-Myc as a control) were co-transfected into HEK293 cells for 24 h and then treated with IFN-α (1,000 U/mL) for 30 min. IP was conducted with an anti-Myc antibody and subsequently IB with an HA-tag antibody. ( E ) HEK293 cells were first transfected with the empty vector (pCMV-Myc) or Myc-KPNA1 for 24 h and then infected with TMUV (MOI = 5) or not for another 24 h. After being treated with IFN-α (500 U/mL) for 30 min, co-IP analysis was performed with anti-Myc antibody, subsequently IB with TMUV-NS5 antibody. ( F ) HeLa cells were transfected with Vec (HA) or HA-NS5 plasmids, and at 24 h post-transfection, cells were treated with 1,000 U/mL IFN-α for 30 min. Then, IFA was performed with anti-HA and KPNA1 antibodies. ( G ) Vec (HA) or HA-NS5 were co-transfected with Myc-KPNA1 into HEK293 cells, and at 24 h post-transfection, cells were treated with IFN-α (1,000 U/mL) for 30 min. IP was performed with an anti-Myc antibody and subsequently IB with KPNB1 antibody. ( H ) HEK293 cells, which were co-transfected with HA-NS5 and Myc-KPNA1 for 24 h, were untreated and treated with IFN-α (1,000 U/mL) for 30 min, and IP was conducted with anti-Myc antibody, subsequently IB with HA-tag antibody. ( I ) HEK293 cells were transfected with Myc-KPNA1 or Vec (Myc) as a control, then infected with TMUV (MOI = 1) for 24 h. After treating the cells with IFN-α (500 U/mL) for 6 h, the ISGs (ISG15, ISG56, Mx1, OAS1) mRNA expressions were measured by qRT-PCR. ( J ) After transfection with different doses of Myc-KPNA1 plasmid, HEK293 cells were inoculated with TMUV (MOI = 1) for another 24 h. Then, the cells were treated with IFN-α (500 U/ml) for 6 h, and TMUV replication levels were detected by qRT-PCR. Density analysis of protein levels was quantified by ImageJ software. Data were presented as the means ± SEM, and comparisons between two groups were performed using an unpaired two-tailed Student’s t test. * P < 0.05, ** P < 0.01 and no significance (n.s). All experiments were independently repeated three times. IFN-α used for treating HEK293 and HeLa cells is a human source.

    Journal: Journal of Virology

    Article Title: Identifying a novel region in the Tembusu virus NS5 protein antagonizing type I interferon signaling

    doi: 10.1128/jvi.01310-25

    Figure Lengend Snippet: TMUV-NS5 interacts with KPNAs, resulting in the reduction of pSTAT1 nuclear traffic. ( A ) HeLa and HEK293 cells were co-transfected with the indicated plasmids of Vec (HA), HA-NS5, and Myc-KPNA1. At 24 h post-transfection, cells were treated with 1,000 U/mL IFN-α for 30 min, and then IP was performed with an anti-Myc antibody and subsequently IB with the pSTAT1 antibody. ( B ) HeLa cells were transfected with the HA-NS5 plasmid or Vec (HA). Twenty-four hours after transfection, cells were stimulated with IFN-α (1,000 U/mL) for 30 min, and the expression levels of endogenous KPNA1, KPNA2, KPNA3, KPNA4, KPNA5, and KPNA6 were analyzed by WB. ( C ) HeLa cells were treated with IFN-α (1,000 U/mL) for 30 min in the presence or absence of HA-NS5, and IP was conducted with anti-HA antibodies, subsequently IB with KPNA1, KPNA2, KPNA3, KPNA4, KPNA5, and KPNA6 antibodies. An empty vector of pCAGGS-HA was used as a control. ( D ) HA-NS5 and Myc-KPNA1 (with the empty vector of pCMV-Myc as a control) were co-transfected into HEK293 cells for 24 h and then treated with IFN-α (1,000 U/mL) for 30 min. IP was conducted with an anti-Myc antibody and subsequently IB with an HA-tag antibody. ( E ) HEK293 cells were first transfected with the empty vector (pCMV-Myc) or Myc-KPNA1 for 24 h and then infected with TMUV (MOI = 5) or not for another 24 h. After being treated with IFN-α (500 U/mL) for 30 min, co-IP analysis was performed with anti-Myc antibody, subsequently IB with TMUV-NS5 antibody. ( F ) HeLa cells were transfected with Vec (HA) or HA-NS5 plasmids, and at 24 h post-transfection, cells were treated with 1,000 U/mL IFN-α for 30 min. Then, IFA was performed with anti-HA and KPNA1 antibodies. ( G ) Vec (HA) or HA-NS5 were co-transfected with Myc-KPNA1 into HEK293 cells, and at 24 h post-transfection, cells were treated with IFN-α (1,000 U/mL) for 30 min. IP was performed with an anti-Myc antibody and subsequently IB with KPNB1 antibody. ( H ) HEK293 cells, which were co-transfected with HA-NS5 and Myc-KPNA1 for 24 h, were untreated and treated with IFN-α (1,000 U/mL) for 30 min, and IP was conducted with anti-Myc antibody, subsequently IB with HA-tag antibody. ( I ) HEK293 cells were transfected with Myc-KPNA1 or Vec (Myc) as a control, then infected with TMUV (MOI = 1) for 24 h. After treating the cells with IFN-α (500 U/mL) for 6 h, the ISGs (ISG15, ISG56, Mx1, OAS1) mRNA expressions were measured by qRT-PCR. ( J ) After transfection with different doses of Myc-KPNA1 plasmid, HEK293 cells were inoculated with TMUV (MOI = 1) for another 24 h. Then, the cells were treated with IFN-α (500 U/ml) for 6 h, and TMUV replication levels were detected by qRT-PCR. Density analysis of protein levels was quantified by ImageJ software. Data were presented as the means ± SEM, and comparisons between two groups were performed using an unpaired two-tailed Student’s t test. * P < 0.05, ** P < 0.01 and no significance (n.s). All experiments were independently repeated three times. IFN-α used for treating HEK293 and HeLa cells is a human source.

    Article Snippet: Rabbit polyclonal antibodies against KPNA1, KPNA2, KPNA3, KPNA4, KPNA5, KPNA6, KPNB1, JAK1, IFNAR1, and IRF9 were purchased from Abclonal Technology.

    Techniques: Transfection, Plasmid Preparation, Expressing, Control, Infection, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Software, Two Tailed Test

    KPNA3 interacts with the NLS of NPAT. (A) MCF-7 cells treated with or without ivermectin (25 μM) were stained with phalloidin for actin and NPAT antibody for HLB. Bar: 10 μm. The insets show the magnification of the boxed region (scale bar, 5 μm). The numbers of HLB foci per cell were quantified as violin plots (DMSO: n = 126; ivermectin: n = 132). ****: P < 0.0001. (B) MCF-7 cells treated with or without ivermectin (25 μM) were subjected to qPCR analysis for transcription levels of the indicated RD histone genes and two control genes. Data in the bar graphs represent the means ± SEM values of the relative transcription levels for three independent experiments. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant. (C) Confocal images of 293T-GFP-NPAT cells treated with or without ivermectin (25 μM). Bar: 10 μm. The insets show the magnification of the boxed region (scale bar, 5 μm). The GFP-NPAT foci per cell were quantified as violin plots (DMSO: n = 85; ivermectin: n = 73). ****: P < 0.0001. (D) Coimmunoprecipitation of FLAG-NPAT and HA-tagged KPNA family members. (E) Schematic diagram of NPAT and its truncation mutants. (F) Coimmunoprecipitation of FLAG-tagged NPAT truncation mutants and GFP-KPNA3. (G) Schematic diagram of the NPAT C-region and its ΔNLS deletion mutant (Δ1,368–1,405). (H) Confocal images of representative MCF-7 cells expressing the GFP-NPAT C-region and its NLS deletion mutant. Scale bar: 5 μm. Data in the bar graphs represent the means ± SD values of the ratio of nuclear GFP signal of WT or NLS deletion mutant for three independent experiments. ****: P < 0.0001. (I) Coimmunoprecipitation of the FLAG-tagged NPAT C-region or its ΔNLS deletion mutant and HA-KPNA3. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: KPNA3 regulates histone locus body formation by modulating condensation and nuclear import of NPAT

    doi: 10.1083/jcb.202401036

    Figure Lengend Snippet: KPNA3 interacts with the NLS of NPAT. (A) MCF-7 cells treated with or without ivermectin (25 μM) were stained with phalloidin for actin and NPAT antibody for HLB. Bar: 10 μm. The insets show the magnification of the boxed region (scale bar, 5 μm). The numbers of HLB foci per cell were quantified as violin plots (DMSO: n = 126; ivermectin: n = 132). ****: P < 0.0001. (B) MCF-7 cells treated with or without ivermectin (25 μM) were subjected to qPCR analysis for transcription levels of the indicated RD histone genes and two control genes. Data in the bar graphs represent the means ± SEM values of the relative transcription levels for three independent experiments. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant. (C) Confocal images of 293T-GFP-NPAT cells treated with or without ivermectin (25 μM). Bar: 10 μm. The insets show the magnification of the boxed region (scale bar, 5 μm). The GFP-NPAT foci per cell were quantified as violin plots (DMSO: n = 85; ivermectin: n = 73). ****: P < 0.0001. (D) Coimmunoprecipitation of FLAG-NPAT and HA-tagged KPNA family members. (E) Schematic diagram of NPAT and its truncation mutants. (F) Coimmunoprecipitation of FLAG-tagged NPAT truncation mutants and GFP-KPNA3. (G) Schematic diagram of the NPAT C-region and its ΔNLS deletion mutant (Δ1,368–1,405). (H) Confocal images of representative MCF-7 cells expressing the GFP-NPAT C-region and its NLS deletion mutant. Scale bar: 5 μm. Data in the bar graphs represent the means ± SD values of the ratio of nuclear GFP signal of WT or NLS deletion mutant for three independent experiments. ****: P < 0.0001. (I) Coimmunoprecipitation of the FLAG-tagged NPAT C-region or its ΔNLS deletion mutant and HA-KPNA3. Source data are available for this figure: .

    Article Snippet: NPAT C-SIF deletion, NLS deletion, and KPNA3 ARM3–5 deletion mutants were constructed using 2×MultiF Seamless Assembly Mix (RK21020; Abclonal) according to the manufacturer’s protocol.

    Techniques: Staining, Control, Mutagenesis, Expressing

    KPNA3 determines the nuclear import of NPAT and HLB formation. (A) Cytoplasmic and nuclear fractions were prepared from MCF-7 cells transfected with control or KPNA3 siRNA and then subjected to western blot analysis for examination of NPAT levels. Data in the bar graphs represent the means ± SEM values of the ratio of densities for three independent experiments. **: P < 0.01, ns: not significant. (B) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with control or KPNA3 siRNA for 48 h. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The endogenous NPAT foci per cell were quantified (control siRNA: n = 125; KPNA3 siRNA: n = 101). ****: P < 0.0001. (C) 293T cells stably expressing GFP-NPAT were transfected with control or KPNA3 siRNA. Cytoplasmic and nuclear fractions were prepared and subjected to western blot analysis for examination of GFP-NPAT levels. Data in the bar graphs represent the means ± SEM values of the ratio of densities for three independent experiments. **: P < 0.01, ns: not significant. (D) Confocal images of the 293T-GFP-NPAT cells transfected with control or KPNA3 siRNA. Bar: 10 μm. The insets show the magnification of the boxed region (scale bar, 5 μm). The GFP-NPAT foci per cell were quantified (control siRNA: n = 83; KPNA3 siRNA: n = 65). ****: P < 0.0001. (E) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with control siRNA or cells transfected with KPNA3 siRNA for 48 h and treated with ivermectin (25 µM). Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The ratio of cells with NPAT foci in cytoplasm was quantified and data in the bar graphs represent the means ± SD value for three independent experiments. **: P < 0.01. The nuclear NPAT foci per cell were quantified (control siRNA: n = 226; KPNA3 siRNA: n = 158). ****: P < 0.0001. (F) MCF-7 cells transfected with control or KPNA3 siRNA were subject to qPCR analysis for transcription levels of the indicated RD histone genes and two control genes. Data in the bar graphs represent the means ± SEM values of the relative transcription levels for three independent experiments. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant. (G) Control or KPNA3 siRNA transfected MCF-7 cells were subjected to colony formation assays and quantified. Data in the bar graphs represent the means ± SD values of the clone numbers for three independent experiments. **: P < 0.01. (H) CCK8 assays were used to assess cell proliferation for MCF-7 cells transfected with control or KPNA3 siRNA. Data in the bar graphs represent the means ± SD values of the OD450 absorption for three independent experiments. ****: P < 0.0001. (I) A model of KPNA3-mediated nuclear import of NPAT as essential for HLB formation and RD-histone gene transcription. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: KPNA3 regulates histone locus body formation by modulating condensation and nuclear import of NPAT

    doi: 10.1083/jcb.202401036

    Figure Lengend Snippet: KPNA3 determines the nuclear import of NPAT and HLB formation. (A) Cytoplasmic and nuclear fractions were prepared from MCF-7 cells transfected with control or KPNA3 siRNA and then subjected to western blot analysis for examination of NPAT levels. Data in the bar graphs represent the means ± SEM values of the ratio of densities for three independent experiments. **: P < 0.01, ns: not significant. (B) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with control or KPNA3 siRNA for 48 h. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The endogenous NPAT foci per cell were quantified (control siRNA: n = 125; KPNA3 siRNA: n = 101). ****: P < 0.0001. (C) 293T cells stably expressing GFP-NPAT were transfected with control or KPNA3 siRNA. Cytoplasmic and nuclear fractions were prepared and subjected to western blot analysis for examination of GFP-NPAT levels. Data in the bar graphs represent the means ± SEM values of the ratio of densities for three independent experiments. **: P < 0.01, ns: not significant. (D) Confocal images of the 293T-GFP-NPAT cells transfected with control or KPNA3 siRNA. Bar: 10 μm. The insets show the magnification of the boxed region (scale bar, 5 μm). The GFP-NPAT foci per cell were quantified (control siRNA: n = 83; KPNA3 siRNA: n = 65). ****: P < 0.0001. (E) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with control siRNA or cells transfected with KPNA3 siRNA for 48 h and treated with ivermectin (25 µM). Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The ratio of cells with NPAT foci in cytoplasm was quantified and data in the bar graphs represent the means ± SD value for three independent experiments. **: P < 0.01. The nuclear NPAT foci per cell were quantified (control siRNA: n = 226; KPNA3 siRNA: n = 158). ****: P < 0.0001. (F) MCF-7 cells transfected with control or KPNA3 siRNA were subject to qPCR analysis for transcription levels of the indicated RD histone genes and two control genes. Data in the bar graphs represent the means ± SEM values of the relative transcription levels for three independent experiments. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant. (G) Control or KPNA3 siRNA transfected MCF-7 cells were subjected to colony formation assays and quantified. Data in the bar graphs represent the means ± SD values of the clone numbers for three independent experiments. **: P < 0.01. (H) CCK8 assays were used to assess cell proliferation for MCF-7 cells transfected with control or KPNA3 siRNA. Data in the bar graphs represent the means ± SD values of the OD450 absorption for three independent experiments. ****: P < 0.0001. (I) A model of KPNA3-mediated nuclear import of NPAT as essential for HLB formation and RD-histone gene transcription. Source data are available for this figure: .

    Article Snippet: NPAT C-SIF deletion, NLS deletion, and KPNA3 ARM3–5 deletion mutants were constructed using 2×MultiF Seamless Assembly Mix (RK21020; Abclonal) according to the manufacturer’s protocol.

    Techniques: Transfection, Control, Western Blot, Immunofluorescence, Staining, Stable Transfection, Expressing

    Ivermectin treatment reduces the nuclear import of NPAT. (A) Western blot analysis of endogenous NPAT expression in MCF-7 cells transfected with control or NPAT siRNA. (B) Cytoplasmic and nuclear fractions were prepared from control or ivermectin-treated MCF-7 cells and then subjected to western blot analysis to examine NPAT levels. Data in the bar graphs represent the means ± SEM values of the ratio of densities for three independent experiments. **: P < 0.01, ns: not significant. (C) Immunofluorescence staining of endogenous NPAT in MCF-7 cells treated with 50 µM ivermectin. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The ratio of cells with NPAT foci in cytoplasm was quantified and data in the bar graphs represent the means ± SD value for three independent experiments. **: P < 0.01. The nuclear NPAT foci per cell were quantified (control: n = 170; ivermectin: n = 166). ****: P < 0.0001. (D) KPNA3 siRNA reduced the expression levels of endogenous KPNA3. (E) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with control or KPNA3 siRNA for 72 h. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The ratio of cells with NPAT foci in cytoplasm was quantified and data in the bar graphs represent the means ± SD value for three independent experiments. **: P < 0.01. The nuclear NPAT foci per cell were quantified (control siRNA: n = 160; KPNA3 siRNA: n = 159). ****: P < 0.0001. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: KPNA3 regulates histone locus body formation by modulating condensation and nuclear import of NPAT

    doi: 10.1083/jcb.202401036

    Figure Lengend Snippet: Ivermectin treatment reduces the nuclear import of NPAT. (A) Western blot analysis of endogenous NPAT expression in MCF-7 cells transfected with control or NPAT siRNA. (B) Cytoplasmic and nuclear fractions were prepared from control or ivermectin-treated MCF-7 cells and then subjected to western blot analysis to examine NPAT levels. Data in the bar graphs represent the means ± SEM values of the ratio of densities for three independent experiments. **: P < 0.01, ns: not significant. (C) Immunofluorescence staining of endogenous NPAT in MCF-7 cells treated with 50 µM ivermectin. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The ratio of cells with NPAT foci in cytoplasm was quantified and data in the bar graphs represent the means ± SD value for three independent experiments. **: P < 0.01. The nuclear NPAT foci per cell were quantified (control: n = 170; ivermectin: n = 166). ****: P < 0.0001. (D) KPNA3 siRNA reduced the expression levels of endogenous KPNA3. (E) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with control or KPNA3 siRNA for 72 h. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The ratio of cells with NPAT foci in cytoplasm was quantified and data in the bar graphs represent the means ± SD value for three independent experiments. **: P < 0.01. The nuclear NPAT foci per cell were quantified (control siRNA: n = 160; KPNA3 siRNA: n = 159). ****: P < 0.0001. Source data are available for this figure: .

    Article Snippet: NPAT C-SIF deletion, NLS deletion, and KPNA3 ARM3–5 deletion mutants were constructed using 2×MultiF Seamless Assembly Mix (RK21020; Abclonal) according to the manufacturer’s protocol.

    Techniques: Western Blot, Expressing, Transfection, Control, Immunofluorescence, Staining

    Comparison of NPAT and KPNA3 concentration. DIC images of FLAG-NPAT and FLAG-KPNA3 condensation at a series of protein concentrations (12.5–25 μM). The size of droplets was quantified (NPAT 12.5 μM: n = 51; NPAT 25 μM: n = 38; KPNA3: N/A). ****: P < 0.0001.

    Journal: The Journal of Cell Biology

    Article Title: KPNA3 regulates histone locus body formation by modulating condensation and nuclear import of NPAT

    doi: 10.1083/jcb.202401036

    Figure Lengend Snippet: Comparison of NPAT and KPNA3 concentration. DIC images of FLAG-NPAT and FLAG-KPNA3 condensation at a series of protein concentrations (12.5–25 μM). The size of droplets was quantified (NPAT 12.5 μM: n = 51; NPAT 25 μM: n = 38; KPNA3: N/A). ****: P < 0.0001.

    Article Snippet: NPAT C-SIF deletion, NLS deletion, and KPNA3 ARM3–5 deletion mutants were constructed using 2×MultiF Seamless Assembly Mix (RK21020; Abclonal) according to the manufacturer’s protocol.

    Techniques: Comparison, Concentration Assay

    KPNA3 suppresses NPAT condensation. (A) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP (12.5 μM) or KPNA3 (12.5 μM). Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 68; NPAT+KPNA3: n = 60). ****: P < 0.0001. (B) Coimmunoprecipitation of FLAG-tagged NPAT and GFP-NPAT in the presence or absence of Myc-KPNA3. Data in the bar graphs represent the means ± SEM values of the association of FLAG-NPAT and GFP-NPAT. *: P < 0.05. (C) Confocal images of MCF-7 cells co-transfected with GFP-NPAT and FLAG vector or FLAG-KPNA3. Bar: 10 μm. The insets show the magnification of the boxed region (scale bar, 5 μm). (D) Cytoplasmic and nuclear fractions were prepared from MCF-7 cells transfected with FLAG vector or FLAG-KPNA3 and then subjected to western blot analysis for examination of NPAT levels. Data in the bar graphs represent the means ± SEM values of the ratio of densities for three independent experiments. *: P < 0.05, ns: not significant. (E) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with FLAG vector or FLAG-KPNA3. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The numbers of endogenous NPAT foci per cell were quantified (FLAG vector: n = 111; FLAG-KPNA3: n = 87). ****: P < 0.0001. (F) MCF-7 cells transfected with a control vector or FLAG-KPNA3 were subject to qPCR analysis for transcription levels of the indicated RD histone genes and two control genes. Data in the bar graphs represent the means ± SEM values of the relative transcription levels for three independent experiments. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant. (G) Schematic diagram of KPNA3 and its truncation mutants. (H) Coimmunoprecipitation of FLAG-tagged KPNA3 truncation mutants and GFP-NPAT C-region. (I) Schematic diagram of KPNA3 and its ARM 3–5 deletion mutant (ΔARM3–5). (J) Coimmunoprecipitation of FLAG-tagged KPNA3 wild type or the ΔARM3–5 mutant and GFP-NPAT C-region. (K) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP, KPNA3, or KPNA3 ΔARM3–5 mutant at the concentration of 12.5 μM. Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 85; NPAT+KPNA3: n = 44; NPAT+KPNA3 ΔARM3–5: n = 70). ****: P < 0.0001. (L) Schematic diagram of NPAT and its NLS deletion mutant (NPAT-ΔNLS). (M) Confocal images of MCF-7 cells transfected with GFP-NPAT or GFP-NPAT-ΔNLS. Bar: 5 μm. The ratio of cells with cytoplasmic NPAT foci was quantified for three independent experiments. ****: P < 0.0001. (N) A model of KPNA3-mediated decondensation of NPAT. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: KPNA3 regulates histone locus body formation by modulating condensation and nuclear import of NPAT

    doi: 10.1083/jcb.202401036

    Figure Lengend Snippet: KPNA3 suppresses NPAT condensation. (A) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP (12.5 μM) or KPNA3 (12.5 μM). Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 68; NPAT+KPNA3: n = 60). ****: P < 0.0001. (B) Coimmunoprecipitation of FLAG-tagged NPAT and GFP-NPAT in the presence or absence of Myc-KPNA3. Data in the bar graphs represent the means ± SEM values of the association of FLAG-NPAT and GFP-NPAT. *: P < 0.05. (C) Confocal images of MCF-7 cells co-transfected with GFP-NPAT and FLAG vector or FLAG-KPNA3. Bar: 10 μm. The insets show the magnification of the boxed region (scale bar, 5 μm). (D) Cytoplasmic and nuclear fractions were prepared from MCF-7 cells transfected with FLAG vector or FLAG-KPNA3 and then subjected to western blot analysis for examination of NPAT levels. Data in the bar graphs represent the means ± SEM values of the ratio of densities for three independent experiments. *: P < 0.05, ns: not significant. (E) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with FLAG vector or FLAG-KPNA3. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The numbers of endogenous NPAT foci per cell were quantified (FLAG vector: n = 111; FLAG-KPNA3: n = 87). ****: P < 0.0001. (F) MCF-7 cells transfected with a control vector or FLAG-KPNA3 were subject to qPCR analysis for transcription levels of the indicated RD histone genes and two control genes. Data in the bar graphs represent the means ± SEM values of the relative transcription levels for three independent experiments. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant. (G) Schematic diagram of KPNA3 and its truncation mutants. (H) Coimmunoprecipitation of FLAG-tagged KPNA3 truncation mutants and GFP-NPAT C-region. (I) Schematic diagram of KPNA3 and its ARM 3–5 deletion mutant (ΔARM3–5). (J) Coimmunoprecipitation of FLAG-tagged KPNA3 wild type or the ΔARM3–5 mutant and GFP-NPAT C-region. (K) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP, KPNA3, or KPNA3 ΔARM3–5 mutant at the concentration of 12.5 μM. Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 85; NPAT+KPNA3: n = 44; NPAT+KPNA3 ΔARM3–5: n = 70). ****: P < 0.0001. (L) Schematic diagram of NPAT and its NLS deletion mutant (NPAT-ΔNLS). (M) Confocal images of MCF-7 cells transfected with GFP-NPAT or GFP-NPAT-ΔNLS. Bar: 5 μm. The ratio of cells with cytoplasmic NPAT foci was quantified for three independent experiments. ****: P < 0.0001. (N) A model of KPNA3-mediated decondensation of NPAT. Source data are available for this figure: .

    Article Snippet: NPAT C-SIF deletion, NLS deletion, and KPNA3 ARM3–5 deletion mutants were constructed using 2×MultiF Seamless Assembly Mix (RK21020; Abclonal) according to the manufacturer’s protocol.

    Techniques: Transfection, Plasmid Preparation, Western Blot, Immunofluorescence, Staining, Control, Mutagenesis, Concentration Assay

    The binding to KPNA3 suppresses the condensation of NPAT. (A) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with FLAG vector, FLAG-KPNA3, or FLAG-KPNA5. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The numbers of endogenous NPAT foci per cell were quantified (FLAG vector: n = 109; FLAG-KPNA3: n = 102; FLAG-KPNA5: n = 107). ****: P < 0.0001, ns: not significant. (B) Schematic diagram of KPNA3, KPNA5, and KPNA5-3-hybrid mutant. (C) Coimmunoprecipitation of GFP-NPAT C-region and FLAG-tagged KPNA3 or KPNA5-3-hybrid mutant. (D) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with FLAG vector, FLAG-KPNA3, FLAG-KPNA5, or FLAG-KPNA5-3-hybrid. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The numbers of endogenous NPAT foci per cell were quantified (FLAG vector: n = 104; FLAG-KPNA3: n = 92; FLAG-KPNA5: n = 95; FLAG-KPNA5-3-hybrid: n = 87). ****: P < 0.0001, ns: not significant. (E) Schematic diagram of NPAT, KPNA3, and NPAT-KPNA3 fusion mutant. (F) Western blot analysis of GFP-NPAT and GFP-NPAT-KPNA3 fusion mutant expression in MCF-7 cells. (G) Confocal images of MCF-7 cells transfected with GFP-NPAT or GFP-NPAT-KPNA3 fusion. Bar: 10 μm. The ratio of cells with nuclear NPAT foci was quantified for three independent experiments. ***: P < 0.001. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: KPNA3 regulates histone locus body formation by modulating condensation and nuclear import of NPAT

    doi: 10.1083/jcb.202401036

    Figure Lengend Snippet: The binding to KPNA3 suppresses the condensation of NPAT. (A) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with FLAG vector, FLAG-KPNA3, or FLAG-KPNA5. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The numbers of endogenous NPAT foci per cell were quantified (FLAG vector: n = 109; FLAG-KPNA3: n = 102; FLAG-KPNA5: n = 107). ****: P < 0.0001, ns: not significant. (B) Schematic diagram of KPNA3, KPNA5, and KPNA5-3-hybrid mutant. (C) Coimmunoprecipitation of GFP-NPAT C-region and FLAG-tagged KPNA3 or KPNA5-3-hybrid mutant. (D) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with FLAG vector, FLAG-KPNA3, FLAG-KPNA5, or FLAG-KPNA5-3-hybrid. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The numbers of endogenous NPAT foci per cell were quantified (FLAG vector: n = 104; FLAG-KPNA3: n = 92; FLAG-KPNA5: n = 95; FLAG-KPNA5-3-hybrid: n = 87). ****: P < 0.0001, ns: not significant. (E) Schematic diagram of NPAT, KPNA3, and NPAT-KPNA3 fusion mutant. (F) Western blot analysis of GFP-NPAT and GFP-NPAT-KPNA3 fusion mutant expression in MCF-7 cells. (G) Confocal images of MCF-7 cells transfected with GFP-NPAT or GFP-NPAT-KPNA3 fusion. Bar: 10 μm. The ratio of cells with nuclear NPAT foci was quantified for three independent experiments. ***: P < 0.001. Source data are available for this figure: .

    Article Snippet: NPAT C-SIF deletion, NLS deletion, and KPNA3 ARM3–5 deletion mutants were constructed using 2×MultiF Seamless Assembly Mix (RK21020; Abclonal) according to the manufacturer’s protocol.

    Techniques: Binding Assay, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Expressing

    Steric binding of KPNA3 interferes with the self-association and condensation of NPAT. (A) Coimmunoprecipitation of FLAG-tagged C-region and GFP-M-region in the presence or absence of GFP-KPNA3. (B) Coimmunoprecipitation of FLAG-tagged C-region or its ΔNLS mutant and GFP-M-region. (C) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP, KPNA3, or KPNA3 ARM 3–5 mutants at a concentration of 12.5 μM. Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 62; NPAT+KPNA3: n = 56; NPAT+KPNA3 ARM3–5: n = 85). ****: P < 0.0001, ns: not significant. (D) Schematic diagram of KPNA3, ARM 3–5 and GFP-ARM 3–5. (E) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP, FLAG-KPNA3 ARM 3–5, or GFP-KPNA3 ARM 3–5 mutant at a concentration of 12.5 μM. Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 78; NPAT+myc-KPNA3 ARM3–5: n = 74; NPAT+GFP-KPNA3 ARM3–5: n = 53). **: P < 0.01. (F) Coimmunoprecipitation of FLAG-tagged C-region and GFP-M-region in the presence of Myc-KPNA3, Myc-AMR 3–5, or GFP-ARM 3–5. (G) Schematic diagram of KPNA3, ARM 1–5, and ARM 3–10. (H) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP, FLAG-KPNA3, FLAG-ARM 1–5, or FLAG-ARM 3–10 mutant at a concentration of 12.5 μM. Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 74; NPAT+KPNA3: n = 80; NPAT+KPNA3 ARM1-5: n = 97; NPAT+KPNA3 ARM3-10: n = 110). ****: P < 0.0001. (I) Coimmunoprecipitation of FLAG-tagged C-region and GFP-M-region in the presence of GFP, Myc-KPNA3, Myc-ARM 1–5, or Myc-ARM 3–10 mutant. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: KPNA3 regulates histone locus body formation by modulating condensation and nuclear import of NPAT

    doi: 10.1083/jcb.202401036

    Figure Lengend Snippet: Steric binding of KPNA3 interferes with the self-association and condensation of NPAT. (A) Coimmunoprecipitation of FLAG-tagged C-region and GFP-M-region in the presence or absence of GFP-KPNA3. (B) Coimmunoprecipitation of FLAG-tagged C-region or its ΔNLS mutant and GFP-M-region. (C) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP, KPNA3, or KPNA3 ARM 3–5 mutants at a concentration of 12.5 μM. Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 62; NPAT+KPNA3: n = 56; NPAT+KPNA3 ARM3–5: n = 85). ****: P < 0.0001, ns: not significant. (D) Schematic diagram of KPNA3, ARM 3–5 and GFP-ARM 3–5. (E) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP, FLAG-KPNA3 ARM 3–5, or GFP-KPNA3 ARM 3–5 mutant at a concentration of 12.5 μM. Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 78; NPAT+myc-KPNA3 ARM3–5: n = 74; NPAT+GFP-KPNA3 ARM3–5: n = 53). **: P < 0.01. (F) Coimmunoprecipitation of FLAG-tagged C-region and GFP-M-region in the presence of Myc-KPNA3, Myc-AMR 3–5, or GFP-ARM 3–5. (G) Schematic diagram of KPNA3, ARM 1–5, and ARM 3–10. (H) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP, FLAG-KPNA3, FLAG-ARM 1–5, or FLAG-ARM 3–10 mutant at a concentration of 12.5 μM. Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 74; NPAT+KPNA3: n = 80; NPAT+KPNA3 ARM1-5: n = 97; NPAT+KPNA3 ARM3-10: n = 110). ****: P < 0.0001. (I) Coimmunoprecipitation of FLAG-tagged C-region and GFP-M-region in the presence of GFP, Myc-KPNA3, Myc-ARM 1–5, or Myc-ARM 3–10 mutant. Source data are available for this figure: .

    Article Snippet: NPAT C-SIF deletion, NLS deletion, and KPNA3 ARM3–5 deletion mutants were constructed using 2×MultiF Seamless Assembly Mix (RK21020; Abclonal) according to the manufacturer’s protocol.

    Techniques: Binding Assay, Mutagenesis, Concentration Assay

    Schematic model for KPNA3-mediated NPAT decondensation and HLB remodeling. We found that KPNA3 determines the nuclear import of NPAT by interacting with the C-terminus NLS region. A C-SIF region proximal to NLS is essential for mediating the self-association of NPAT to drive the condensation of NPAT in nuclei. In the cytoplasm, NLS-mediated KPNA3 association sterically blocks the C-SIF motif-dependent NPAT self-association, which ensures the nuclear import of NPAT and prevents aberrant cytoplasmic HLB formation.

    Journal: The Journal of Cell Biology

    Article Title: KPNA3 regulates histone locus body formation by modulating condensation and nuclear import of NPAT

    doi: 10.1083/jcb.202401036

    Figure Lengend Snippet: Schematic model for KPNA3-mediated NPAT decondensation and HLB remodeling. We found that KPNA3 determines the nuclear import of NPAT by interacting with the C-terminus NLS region. A C-SIF region proximal to NLS is essential for mediating the self-association of NPAT to drive the condensation of NPAT in nuclei. In the cytoplasm, NLS-mediated KPNA3 association sterically blocks the C-SIF motif-dependent NPAT self-association, which ensures the nuclear import of NPAT and prevents aberrant cytoplasmic HLB formation.

    Article Snippet: NPAT C-SIF deletion, NLS deletion, and KPNA3 ARM3–5 deletion mutants were constructed using 2×MultiF Seamless Assembly Mix (RK21020; Abclonal) according to the manufacturer’s protocol.

    Techniques:

    KPNA3 interacts with the NLS of NPAT. (A) MCF-7 cells treated with or without ivermectin (25 μM) were stained with phalloidin for actin and NPAT antibody for HLB. Bar: 10 μm. The insets show the magnification of the boxed region (scale bar, 5 μm). The numbers of HLB foci per cell were quantified as violin plots (DMSO: n = 126; ivermectin: n = 132). ****: P < 0.0001. (B) MCF-7 cells treated with or without ivermectin (25 μM) were subjected to qPCR analysis for transcription levels of the indicated RD histone genes and two control genes. Data in the bar graphs represent the means ± SEM values of the relative transcription levels for three independent experiments. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant. (C) Confocal images of 293T-GFP-NPAT cells treated with or without ivermectin (25 μM). Bar: 10 μm. The insets show the magnification of the boxed region (scale bar, 5 μm). The GFP-NPAT foci per cell were quantified as violin plots (DMSO: n = 85; ivermectin: n = 73). ****: P < 0.0001. (D) Coimmunoprecipitation of FLAG-NPAT and HA-tagged KPNA family members. (E) Schematic diagram of NPAT and its truncation mutants. (F) Coimmunoprecipitation of FLAG-tagged NPAT truncation mutants and GFP-KPNA3. (G) Schematic diagram of the NPAT C-region and its ΔNLS deletion mutant (Δ1,368–1,405). (H) Confocal images of representative MCF-7 cells expressing the GFP-NPAT C-region and its NLS deletion mutant. Scale bar: 5 μm. Data in the bar graphs represent the means ± SD values of the ratio of nuclear GFP signal of WT or NLS deletion mutant for three independent experiments. ****: P < 0.0001. (I) Coimmunoprecipitation of the FLAG-tagged NPAT C-region or its ΔNLS deletion mutant and HA-KPNA3. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: KPNA3 regulates histone locus body formation by modulating condensation and nuclear import of NPAT

    doi: 10.1083/jcb.202401036

    Figure Lengend Snippet: KPNA3 interacts with the NLS of NPAT. (A) MCF-7 cells treated with or without ivermectin (25 μM) were stained with phalloidin for actin and NPAT antibody for HLB. Bar: 10 μm. The insets show the magnification of the boxed region (scale bar, 5 μm). The numbers of HLB foci per cell were quantified as violin plots (DMSO: n = 126; ivermectin: n = 132). ****: P < 0.0001. (B) MCF-7 cells treated with or without ivermectin (25 μM) were subjected to qPCR analysis for transcription levels of the indicated RD histone genes and two control genes. Data in the bar graphs represent the means ± SEM values of the relative transcription levels for three independent experiments. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant. (C) Confocal images of 293T-GFP-NPAT cells treated with or without ivermectin (25 μM). Bar: 10 μm. The insets show the magnification of the boxed region (scale bar, 5 μm). The GFP-NPAT foci per cell were quantified as violin plots (DMSO: n = 85; ivermectin: n = 73). ****: P < 0.0001. (D) Coimmunoprecipitation of FLAG-NPAT and HA-tagged KPNA family members. (E) Schematic diagram of NPAT and its truncation mutants. (F) Coimmunoprecipitation of FLAG-tagged NPAT truncation mutants and GFP-KPNA3. (G) Schematic diagram of the NPAT C-region and its ΔNLS deletion mutant (Δ1,368–1,405). (H) Confocal images of representative MCF-7 cells expressing the GFP-NPAT C-region and its NLS deletion mutant. Scale bar: 5 μm. Data in the bar graphs represent the means ± SD values of the ratio of nuclear GFP signal of WT or NLS deletion mutant for three independent experiments. ****: P < 0.0001. (I) Coimmunoprecipitation of the FLAG-tagged NPAT C-region or its ΔNLS deletion mutant and HA-KPNA3. Source data are available for this figure: .

    Article Snippet: 1 × 10 3 MCF-7 cells were seeded in 96-well plates in triplicate and treated with KPNA3 siRNA for 72 h. Cell growth was measured using a Cell Counting Kit-8 (CCK-8) (HY-K0301; MCE) every 24 h. Briefly, MCF-7 cells were washed twice with PBS and mixed with a reaction solution containing CCK8.

    Techniques: Staining, Control, Mutagenesis, Expressing

    KPNA3 determines the nuclear import of NPAT and HLB formation. (A) Cytoplasmic and nuclear fractions were prepared from MCF-7 cells transfected with control or KPNA3 siRNA and then subjected to western blot analysis for examination of NPAT levels. Data in the bar graphs represent the means ± SEM values of the ratio of densities for three independent experiments. **: P < 0.01, ns: not significant. (B) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with control or KPNA3 siRNA for 48 h. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The endogenous NPAT foci per cell were quantified (control siRNA: n = 125; KPNA3 siRNA: n = 101). ****: P < 0.0001. (C) 293T cells stably expressing GFP-NPAT were transfected with control or KPNA3 siRNA. Cytoplasmic and nuclear fractions were prepared and subjected to western blot analysis for examination of GFP-NPAT levels. Data in the bar graphs represent the means ± SEM values of the ratio of densities for three independent experiments. **: P < 0.01, ns: not significant. (D) Confocal images of the 293T-GFP-NPAT cells transfected with control or KPNA3 siRNA. Bar: 10 μm. The insets show the magnification of the boxed region (scale bar, 5 μm). The GFP-NPAT foci per cell were quantified (control siRNA: n = 83; KPNA3 siRNA: n = 65). ****: P < 0.0001. (E) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with control siRNA or cells transfected with KPNA3 siRNA for 48 h and treated with ivermectin (25 µM). Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The ratio of cells with NPAT foci in cytoplasm was quantified and data in the bar graphs represent the means ± SD value for three independent experiments. **: P < 0.01. The nuclear NPAT foci per cell were quantified (control siRNA: n = 226; KPNA3 siRNA: n = 158). ****: P < 0.0001. (F) MCF-7 cells transfected with control or KPNA3 siRNA were subject to qPCR analysis for transcription levels of the indicated RD histone genes and two control genes. Data in the bar graphs represent the means ± SEM values of the relative transcription levels for three independent experiments. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant. (G) Control or KPNA3 siRNA transfected MCF-7 cells were subjected to colony formation assays and quantified. Data in the bar graphs represent the means ± SD values of the clone numbers for three independent experiments. **: P < 0.01. (H) CCK8 assays were used to assess cell proliferation for MCF-7 cells transfected with control or KPNA3 siRNA. Data in the bar graphs represent the means ± SD values of the OD450 absorption for three independent experiments. ****: P < 0.0001. (I) A model of KPNA3-mediated nuclear import of NPAT as essential for HLB formation and RD-histone gene transcription. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: KPNA3 regulates histone locus body formation by modulating condensation and nuclear import of NPAT

    doi: 10.1083/jcb.202401036

    Figure Lengend Snippet: KPNA3 determines the nuclear import of NPAT and HLB formation. (A) Cytoplasmic and nuclear fractions were prepared from MCF-7 cells transfected with control or KPNA3 siRNA and then subjected to western blot analysis for examination of NPAT levels. Data in the bar graphs represent the means ± SEM values of the ratio of densities for three independent experiments. **: P < 0.01, ns: not significant. (B) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with control or KPNA3 siRNA for 48 h. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The endogenous NPAT foci per cell were quantified (control siRNA: n = 125; KPNA3 siRNA: n = 101). ****: P < 0.0001. (C) 293T cells stably expressing GFP-NPAT were transfected with control or KPNA3 siRNA. Cytoplasmic and nuclear fractions were prepared and subjected to western blot analysis for examination of GFP-NPAT levels. Data in the bar graphs represent the means ± SEM values of the ratio of densities for three independent experiments. **: P < 0.01, ns: not significant. (D) Confocal images of the 293T-GFP-NPAT cells transfected with control or KPNA3 siRNA. Bar: 10 μm. The insets show the magnification of the boxed region (scale bar, 5 μm). The GFP-NPAT foci per cell were quantified (control siRNA: n = 83; KPNA3 siRNA: n = 65). ****: P < 0.0001. (E) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with control siRNA or cells transfected with KPNA3 siRNA for 48 h and treated with ivermectin (25 µM). Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The ratio of cells with NPAT foci in cytoplasm was quantified and data in the bar graphs represent the means ± SD value for three independent experiments. **: P < 0.01. The nuclear NPAT foci per cell were quantified (control siRNA: n = 226; KPNA3 siRNA: n = 158). ****: P < 0.0001. (F) MCF-7 cells transfected with control or KPNA3 siRNA were subject to qPCR analysis for transcription levels of the indicated RD histone genes and two control genes. Data in the bar graphs represent the means ± SEM values of the relative transcription levels for three independent experiments. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant. (G) Control or KPNA3 siRNA transfected MCF-7 cells were subjected to colony formation assays and quantified. Data in the bar graphs represent the means ± SD values of the clone numbers for three independent experiments. **: P < 0.01. (H) CCK8 assays were used to assess cell proliferation for MCF-7 cells transfected with control or KPNA3 siRNA. Data in the bar graphs represent the means ± SD values of the OD450 absorption for three independent experiments. ****: P < 0.0001. (I) A model of KPNA3-mediated nuclear import of NPAT as essential for HLB formation and RD-histone gene transcription. Source data are available for this figure: .

    Article Snippet: 1 × 10 3 MCF-7 cells were seeded in 96-well plates in triplicate and treated with KPNA3 siRNA for 72 h. Cell growth was measured using a Cell Counting Kit-8 (CCK-8) (HY-K0301; MCE) every 24 h. Briefly, MCF-7 cells were washed twice with PBS and mixed with a reaction solution containing CCK8.

    Techniques: Transfection, Control, Western Blot, Immunofluorescence, Staining, Stable Transfection, Expressing

    Ivermectin treatment reduces the nuclear import of NPAT. (A) Western blot analysis of endogenous NPAT expression in MCF-7 cells transfected with control or NPAT siRNA. (B) Cytoplasmic and nuclear fractions were prepared from control or ivermectin-treated MCF-7 cells and then subjected to western blot analysis to examine NPAT levels. Data in the bar graphs represent the means ± SEM values of the ratio of densities for three independent experiments. **: P < 0.01, ns: not significant. (C) Immunofluorescence staining of endogenous NPAT in MCF-7 cells treated with 50 µM ivermectin. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The ratio of cells with NPAT foci in cytoplasm was quantified and data in the bar graphs represent the means ± SD value for three independent experiments. **: P < 0.01. The nuclear NPAT foci per cell were quantified (control: n = 170; ivermectin: n = 166). ****: P < 0.0001. (D) KPNA3 siRNA reduced the expression levels of endogenous KPNA3. (E) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with control or KPNA3 siRNA for 72 h. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The ratio of cells with NPAT foci in cytoplasm was quantified and data in the bar graphs represent the means ± SD value for three independent experiments. **: P < 0.01. The nuclear NPAT foci per cell were quantified (control siRNA: n = 160; KPNA3 siRNA: n = 159). ****: P < 0.0001. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: KPNA3 regulates histone locus body formation by modulating condensation and nuclear import of NPAT

    doi: 10.1083/jcb.202401036

    Figure Lengend Snippet: Ivermectin treatment reduces the nuclear import of NPAT. (A) Western blot analysis of endogenous NPAT expression in MCF-7 cells transfected with control or NPAT siRNA. (B) Cytoplasmic and nuclear fractions were prepared from control or ivermectin-treated MCF-7 cells and then subjected to western blot analysis to examine NPAT levels. Data in the bar graphs represent the means ± SEM values of the ratio of densities for three independent experiments. **: P < 0.01, ns: not significant. (C) Immunofluorescence staining of endogenous NPAT in MCF-7 cells treated with 50 µM ivermectin. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The ratio of cells with NPAT foci in cytoplasm was quantified and data in the bar graphs represent the means ± SD value for three independent experiments. **: P < 0.01. The nuclear NPAT foci per cell were quantified (control: n = 170; ivermectin: n = 166). ****: P < 0.0001. (D) KPNA3 siRNA reduced the expression levels of endogenous KPNA3. (E) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with control or KPNA3 siRNA for 72 h. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The ratio of cells with NPAT foci in cytoplasm was quantified and data in the bar graphs represent the means ± SD value for three independent experiments. **: P < 0.01. The nuclear NPAT foci per cell were quantified (control siRNA: n = 160; KPNA3 siRNA: n = 159). ****: P < 0.0001. Source data are available for this figure: .

    Article Snippet: 1 × 10 3 MCF-7 cells were seeded in 96-well plates in triplicate and treated with KPNA3 siRNA for 72 h. Cell growth was measured using a Cell Counting Kit-8 (CCK-8) (HY-K0301; MCE) every 24 h. Briefly, MCF-7 cells were washed twice with PBS and mixed with a reaction solution containing CCK8.

    Techniques: Western Blot, Expressing, Transfection, Control, Immunofluorescence, Staining

    Comparison of NPAT and KPNA3 concentration. DIC images of FLAG-NPAT and FLAG-KPNA3 condensation at a series of protein concentrations (12.5–25 μM). The size of droplets was quantified (NPAT 12.5 μM: n = 51; NPAT 25 μM: n = 38; KPNA3: N/A). ****: P < 0.0001.

    Journal: The Journal of Cell Biology

    Article Title: KPNA3 regulates histone locus body formation by modulating condensation and nuclear import of NPAT

    doi: 10.1083/jcb.202401036

    Figure Lengend Snippet: Comparison of NPAT and KPNA3 concentration. DIC images of FLAG-NPAT and FLAG-KPNA3 condensation at a series of protein concentrations (12.5–25 μM). The size of droplets was quantified (NPAT 12.5 μM: n = 51; NPAT 25 μM: n = 38; KPNA3: N/A). ****: P < 0.0001.

    Article Snippet: 1 × 10 3 MCF-7 cells were seeded in 96-well plates in triplicate and treated with KPNA3 siRNA for 72 h. Cell growth was measured using a Cell Counting Kit-8 (CCK-8) (HY-K0301; MCE) every 24 h. Briefly, MCF-7 cells were washed twice with PBS and mixed with a reaction solution containing CCK8.

    Techniques: Comparison, Concentration Assay

    KPNA3 suppresses NPAT condensation. (A) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP (12.5 μM) or KPNA3 (12.5 μM). Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 68; NPAT+KPNA3: n = 60). ****: P < 0.0001. (B) Coimmunoprecipitation of FLAG-tagged NPAT and GFP-NPAT in the presence or absence of Myc-KPNA3. Data in the bar graphs represent the means ± SEM values of the association of FLAG-NPAT and GFP-NPAT. *: P < 0.05. (C) Confocal images of MCF-7 cells co-transfected with GFP-NPAT and FLAG vector or FLAG-KPNA3. Bar: 10 μm. The insets show the magnification of the boxed region (scale bar, 5 μm). (D) Cytoplasmic and nuclear fractions were prepared from MCF-7 cells transfected with FLAG vector or FLAG-KPNA3 and then subjected to western blot analysis for examination of NPAT levels. Data in the bar graphs represent the means ± SEM values of the ratio of densities for three independent experiments. *: P < 0.05, ns: not significant. (E) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with FLAG vector or FLAG-KPNA3. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The numbers of endogenous NPAT foci per cell were quantified (FLAG vector: n = 111; FLAG-KPNA3: n = 87). ****: P < 0.0001. (F) MCF-7 cells transfected with a control vector or FLAG-KPNA3 were subject to qPCR analysis for transcription levels of the indicated RD histone genes and two control genes. Data in the bar graphs represent the means ± SEM values of the relative transcription levels for three independent experiments. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant. (G) Schematic diagram of KPNA3 and its truncation mutants. (H) Coimmunoprecipitation of FLAG-tagged KPNA3 truncation mutants and GFP-NPAT C-region. (I) Schematic diagram of KPNA3 and its ARM 3–5 deletion mutant (ΔARM3–5). (J) Coimmunoprecipitation of FLAG-tagged KPNA3 wild type or the ΔARM3–5 mutant and GFP-NPAT C-region. (K) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP, KPNA3, or KPNA3 ΔARM3–5 mutant at the concentration of 12.5 μM. Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 85; NPAT+KPNA3: n = 44; NPAT+KPNA3 ΔARM3–5: n = 70). ****: P < 0.0001. (L) Schematic diagram of NPAT and its NLS deletion mutant (NPAT-ΔNLS). (M) Confocal images of MCF-7 cells transfected with GFP-NPAT or GFP-NPAT-ΔNLS. Bar: 5 μm. The ratio of cells with cytoplasmic NPAT foci was quantified for three independent experiments. ****: P < 0.0001. (N) A model of KPNA3-mediated decondensation of NPAT. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: KPNA3 regulates histone locus body formation by modulating condensation and nuclear import of NPAT

    doi: 10.1083/jcb.202401036

    Figure Lengend Snippet: KPNA3 suppresses NPAT condensation. (A) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP (12.5 μM) or KPNA3 (12.5 μM). Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 68; NPAT+KPNA3: n = 60). ****: P < 0.0001. (B) Coimmunoprecipitation of FLAG-tagged NPAT and GFP-NPAT in the presence or absence of Myc-KPNA3. Data in the bar graphs represent the means ± SEM values of the association of FLAG-NPAT and GFP-NPAT. *: P < 0.05. (C) Confocal images of MCF-7 cells co-transfected with GFP-NPAT and FLAG vector or FLAG-KPNA3. Bar: 10 μm. The insets show the magnification of the boxed region (scale bar, 5 μm). (D) Cytoplasmic and nuclear fractions were prepared from MCF-7 cells transfected with FLAG vector or FLAG-KPNA3 and then subjected to western blot analysis for examination of NPAT levels. Data in the bar graphs represent the means ± SEM values of the ratio of densities for three independent experiments. *: P < 0.05, ns: not significant. (E) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with FLAG vector or FLAG-KPNA3. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The numbers of endogenous NPAT foci per cell were quantified (FLAG vector: n = 111; FLAG-KPNA3: n = 87). ****: P < 0.0001. (F) MCF-7 cells transfected with a control vector or FLAG-KPNA3 were subject to qPCR analysis for transcription levels of the indicated RD histone genes and two control genes. Data in the bar graphs represent the means ± SEM values of the relative transcription levels for three independent experiments. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant. (G) Schematic diagram of KPNA3 and its truncation mutants. (H) Coimmunoprecipitation of FLAG-tagged KPNA3 truncation mutants and GFP-NPAT C-region. (I) Schematic diagram of KPNA3 and its ARM 3–5 deletion mutant (ΔARM3–5). (J) Coimmunoprecipitation of FLAG-tagged KPNA3 wild type or the ΔARM3–5 mutant and GFP-NPAT C-region. (K) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP, KPNA3, or KPNA3 ΔARM3–5 mutant at the concentration of 12.5 μM. Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 85; NPAT+KPNA3: n = 44; NPAT+KPNA3 ΔARM3–5: n = 70). ****: P < 0.0001. (L) Schematic diagram of NPAT and its NLS deletion mutant (NPAT-ΔNLS). (M) Confocal images of MCF-7 cells transfected with GFP-NPAT or GFP-NPAT-ΔNLS. Bar: 5 μm. The ratio of cells with cytoplasmic NPAT foci was quantified for three independent experiments. ****: P < 0.0001. (N) A model of KPNA3-mediated decondensation of NPAT. Source data are available for this figure: .

    Article Snippet: 1 × 10 3 MCF-7 cells were seeded in 96-well plates in triplicate and treated with KPNA3 siRNA for 72 h. Cell growth was measured using a Cell Counting Kit-8 (CCK-8) (HY-K0301; MCE) every 24 h. Briefly, MCF-7 cells were washed twice with PBS and mixed with a reaction solution containing CCK8.

    Techniques: Transfection, Plasmid Preparation, Western Blot, Immunofluorescence, Staining, Control, Mutagenesis, Concentration Assay

    The binding to KPNA3 suppresses the condensation of NPAT. (A) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with FLAG vector, FLAG-KPNA3, or FLAG-KPNA5. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The numbers of endogenous NPAT foci per cell were quantified (FLAG vector: n = 109; FLAG-KPNA3: n = 102; FLAG-KPNA5: n = 107). ****: P < 0.0001, ns: not significant. (B) Schematic diagram of KPNA3, KPNA5, and KPNA5-3-hybrid mutant. (C) Coimmunoprecipitation of GFP-NPAT C-region and FLAG-tagged KPNA3 or KPNA5-3-hybrid mutant. (D) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with FLAG vector, FLAG-KPNA3, FLAG-KPNA5, or FLAG-KPNA5-3-hybrid. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The numbers of endogenous NPAT foci per cell were quantified (FLAG vector: n = 104; FLAG-KPNA3: n = 92; FLAG-KPNA5: n = 95; FLAG-KPNA5-3-hybrid: n = 87). ****: P < 0.0001, ns: not significant. (E) Schematic diagram of NPAT, KPNA3, and NPAT-KPNA3 fusion mutant. (F) Western blot analysis of GFP-NPAT and GFP-NPAT-KPNA3 fusion mutant expression in MCF-7 cells. (G) Confocal images of MCF-7 cells transfected with GFP-NPAT or GFP-NPAT-KPNA3 fusion. Bar: 10 μm. The ratio of cells with nuclear NPAT foci was quantified for three independent experiments. ***: P < 0.001. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: KPNA3 regulates histone locus body formation by modulating condensation and nuclear import of NPAT

    doi: 10.1083/jcb.202401036

    Figure Lengend Snippet: The binding to KPNA3 suppresses the condensation of NPAT. (A) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with FLAG vector, FLAG-KPNA3, or FLAG-KPNA5. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The numbers of endogenous NPAT foci per cell were quantified (FLAG vector: n = 109; FLAG-KPNA3: n = 102; FLAG-KPNA5: n = 107). ****: P < 0.0001, ns: not significant. (B) Schematic diagram of KPNA3, KPNA5, and KPNA5-3-hybrid mutant. (C) Coimmunoprecipitation of GFP-NPAT C-region and FLAG-tagged KPNA3 or KPNA5-3-hybrid mutant. (D) Immunofluorescence staining of endogenous NPAT in MCF-7 cells transfected with FLAG vector, FLAG-KPNA3, FLAG-KPNA5, or FLAG-KPNA5-3-hybrid. Scale bar, 10 µm. The insets show the magnification of the boxed region (scale bar, 5 μm). The numbers of endogenous NPAT foci per cell were quantified (FLAG vector: n = 104; FLAG-KPNA3: n = 92; FLAG-KPNA5: n = 95; FLAG-KPNA5-3-hybrid: n = 87). ****: P < 0.0001, ns: not significant. (E) Schematic diagram of NPAT, KPNA3, and NPAT-KPNA3 fusion mutant. (F) Western blot analysis of GFP-NPAT and GFP-NPAT-KPNA3 fusion mutant expression in MCF-7 cells. (G) Confocal images of MCF-7 cells transfected with GFP-NPAT or GFP-NPAT-KPNA3 fusion. Bar: 10 μm. The ratio of cells with nuclear NPAT foci was quantified for three independent experiments. ***: P < 0.001. Source data are available for this figure: .

    Article Snippet: 1 × 10 3 MCF-7 cells were seeded in 96-well plates in triplicate and treated with KPNA3 siRNA for 72 h. Cell growth was measured using a Cell Counting Kit-8 (CCK-8) (HY-K0301; MCE) every 24 h. Briefly, MCF-7 cells were washed twice with PBS and mixed with a reaction solution containing CCK8.

    Techniques: Binding Assay, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Expressing

    Steric binding of KPNA3 interferes with the self-association and condensation of NPAT. (A) Coimmunoprecipitation of FLAG-tagged C-region and GFP-M-region in the presence or absence of GFP-KPNA3. (B) Coimmunoprecipitation of FLAG-tagged C-region or its ΔNLS mutant and GFP-M-region. (C) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP, KPNA3, or KPNA3 ARM 3–5 mutants at a concentration of 12.5 μM. Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 62; NPAT+KPNA3: n = 56; NPAT+KPNA3 ARM3–5: n = 85). ****: P < 0.0001, ns: not significant. (D) Schematic diagram of KPNA3, ARM 3–5 and GFP-ARM 3–5. (E) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP, FLAG-KPNA3 ARM 3–5, or GFP-KPNA3 ARM 3–5 mutant at a concentration of 12.5 μM. Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 78; NPAT+myc-KPNA3 ARM3–5: n = 74; NPAT+GFP-KPNA3 ARM3–5: n = 53). **: P < 0.01. (F) Coimmunoprecipitation of FLAG-tagged C-region and GFP-M-region in the presence of Myc-KPNA3, Myc-AMR 3–5, or GFP-ARM 3–5. (G) Schematic diagram of KPNA3, ARM 1–5, and ARM 3–10. (H) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP, FLAG-KPNA3, FLAG-ARM 1–5, or FLAG-ARM 3–10 mutant at a concentration of 12.5 μM. Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 74; NPAT+KPNA3: n = 80; NPAT+KPNA3 ARM1-5: n = 97; NPAT+KPNA3 ARM3-10: n = 110). ****: P < 0.0001. (I) Coimmunoprecipitation of FLAG-tagged C-region and GFP-M-region in the presence of GFP, Myc-KPNA3, Myc-ARM 1–5, or Myc-ARM 3–10 mutant. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: KPNA3 regulates histone locus body formation by modulating condensation and nuclear import of NPAT

    doi: 10.1083/jcb.202401036

    Figure Lengend Snippet: Steric binding of KPNA3 interferes with the self-association and condensation of NPAT. (A) Coimmunoprecipitation of FLAG-tagged C-region and GFP-M-region in the presence or absence of GFP-KPNA3. (B) Coimmunoprecipitation of FLAG-tagged C-region or its ΔNLS mutant and GFP-M-region. (C) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP, KPNA3, or KPNA3 ARM 3–5 mutants at a concentration of 12.5 μM. Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 62; NPAT+KPNA3: n = 56; NPAT+KPNA3 ARM3–5: n = 85). ****: P < 0.0001, ns: not significant. (D) Schematic diagram of KPNA3, ARM 3–5 and GFP-ARM 3–5. (E) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP, FLAG-KPNA3 ARM 3–5, or GFP-KPNA3 ARM 3–5 mutant at a concentration of 12.5 μM. Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 78; NPAT+myc-KPNA3 ARM3–5: n = 74; NPAT+GFP-KPNA3 ARM3–5: n = 53). **: P < 0.01. (F) Coimmunoprecipitation of FLAG-tagged C-region and GFP-M-region in the presence of Myc-KPNA3, Myc-AMR 3–5, or GFP-ARM 3–5. (G) Schematic diagram of KPNA3, ARM 1–5, and ARM 3–10. (H) DIC images of FLAG-NPAT (12.5 μM) condensation in the presence of GFP, FLAG-KPNA3, FLAG-ARM 1–5, or FLAG-ARM 3–10 mutant at a concentration of 12.5 μM. Scale bar, 10 µm. The size of droplets was quantified (NPAT+GFP: n = 74; NPAT+KPNA3: n = 80; NPAT+KPNA3 ARM1-5: n = 97; NPAT+KPNA3 ARM3-10: n = 110). ****: P < 0.0001. (I) Coimmunoprecipitation of FLAG-tagged C-region and GFP-M-region in the presence of GFP, Myc-KPNA3, Myc-ARM 1–5, or Myc-ARM 3–10 mutant. Source data are available for this figure: .

    Article Snippet: 1 × 10 3 MCF-7 cells were seeded in 96-well plates in triplicate and treated with KPNA3 siRNA for 72 h. Cell growth was measured using a Cell Counting Kit-8 (CCK-8) (HY-K0301; MCE) every 24 h. Briefly, MCF-7 cells were washed twice with PBS and mixed with a reaction solution containing CCK8.

    Techniques: Binding Assay, Mutagenesis, Concentration Assay

    Schematic model for KPNA3-mediated NPAT decondensation and HLB remodeling. We found that KPNA3 determines the nuclear import of NPAT by interacting with the C-terminus NLS region. A C-SIF region proximal to NLS is essential for mediating the self-association of NPAT to drive the condensation of NPAT in nuclei. In the cytoplasm, NLS-mediated KPNA3 association sterically blocks the C-SIF motif-dependent NPAT self-association, which ensures the nuclear import of NPAT and prevents aberrant cytoplasmic HLB formation.

    Journal: The Journal of Cell Biology

    Article Title: KPNA3 regulates histone locus body formation by modulating condensation and nuclear import of NPAT

    doi: 10.1083/jcb.202401036

    Figure Lengend Snippet: Schematic model for KPNA3-mediated NPAT decondensation and HLB remodeling. We found that KPNA3 determines the nuclear import of NPAT by interacting with the C-terminus NLS region. A C-SIF region proximal to NLS is essential for mediating the self-association of NPAT to drive the condensation of NPAT in nuclei. In the cytoplasm, NLS-mediated KPNA3 association sterically blocks the C-SIF motif-dependent NPAT self-association, which ensures the nuclear import of NPAT and prevents aberrant cytoplasmic HLB formation.

    Article Snippet: 1 × 10 3 MCF-7 cells were seeded in 96-well plates in triplicate and treated with KPNA3 siRNA for 72 h. Cell growth was measured using a Cell Counting Kit-8 (CCK-8) (HY-K0301; MCE) every 24 h. Briefly, MCF-7 cells were washed twice with PBS and mixed with a reaction solution containing CCK8.

    Techniques: