Journal: Viruses

Article Title: NXT1, a Novel Influenza A NP Binding Protein, Promotes the Nuclear Export of NP via a CRM1-Dependent Pathway

doi: 10.3390/v8080209

Figure Lengend Snippet: Identification of nuclear transport factor 2 (NTF2)-like export protein 1 (NXT1) as a binding partner of influenza nucleoprotein (NP). ( A ) NP-FLAG or FLAG- non-structural 1 (NS1) protein was immobilized on anti-FLAG agarose beads and co-incubated with HeLa whole cell lysate overnight at 4 °C. The beads were collected, washed, and subjected to western blot (WB) analysis with anti-NXT1, anti-NUP153, anti-NUP98, anti-NUP62, anti-E1B-AP5, anti-CRM1, or anti-FLAG antibodies. Data shown are the results of one representative experiment from two independent trials. Black arrowheads indicate specific binding. H and L indicate the heavy and light chains of the agarose-conjugated anti-FLAG antibody; ( B ) Human embryonic kidney (HEK293T) cells were transfected with NP-FLAG/pCAGGS and HA-NXT1/pCAGGS plasmids for 48 h, lysed, and incubated with anti-FLAG agarose beads overnight at 4 °C. The agarose beads were collected, washed, and subjected to WB analysis with anti- hemagglutinin (HA) and anti-FLAG antibodies. Data shown are the results of one representative experiment from two independent trials; ( C ) HA-NXT1 protein was immobilized on anti-HA agarose beads and co-incubated with purified NP-FLAG protein overnight at 4 °C. The beads were collected, washed, and subjected to WB analysis with anti-FLAG and anti-HA antibodies. Data shown are the results of one representative experiment from three independent trials.

Article Snippet: Antibodies used for analysis were anti-NXT1 rabbit polyclonal antibody (pAb) (Abnova, Taipei, Taiwan), anti-CRM1 mouse monoclonal antibody (mAb) (BD Biosciences, San Jose, CA, USA), anti-NUP153 rat mAb (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-NUP98 rat mAb (Abcam, Cambridge, UK), anti-NUP62 mouse mAb (BD Biosciences), anti-E1B-AP5 (or hnRNP UL1) mouse mAb (Santa Cruz Biotechnology), anti-FLAG M2 mouse mAb (Sigma-Aldrich, Saint Louis, MO, USA), anti-FLAG rabbit mAb (Sigma-Aldrich), anti-HA mouse mAb (Medical and Biological Laboratories, Nagoya, Japan), anti-β-actin mouse mAb (Sigma-Aldrich), anti-WSN (influenza A/WSN/33) rabbit pAb (a gift from Dr. Kazufumi Shimizu, Nihon University School of Medicine, Tokyo, Japan), horseradish-peroxidase (HRP)-conjugated goat anti-mouse IgG (Amersham Biosciences, Uppsala, Sweden), or HRP-conjugated goat anti-rabbit IgG (Amersham Biosciences).

Techniques: Binding Assay, Incubation, Western Blot, Transfection, Purification

Journal: Stem cell research & therapy

Article Title: Nucleoporin 153 deficiency in adult neural stem cells defines a pathological protein-network signature and defective neurogenesis in a mouse model of AD.

doi: 10.1186/s13287-024-03805-1

Figure Lengend Snippet: Fig. 1 (A) Cartoon depicting the experimental plan for the in vitro experiments. (B) Schematic illustrating the main biological processes from Gene ontol ogy analysis of Nup153-protein network in WT-NSCs

Article Snippet: All mice were randomly assigned to receive Nup153 or GFP-control lentiviral particles (1.5 μl; 1 × 109/ml Origene CW305175V: lentiviral particles produced from PS100071 and MR211997L4) that were delivered in the hippocampus by a stereotaxic injection.

Techniques: In Vitro

Journal: Stem cell research & therapy

Article Title: Nucleoporin 153 deficiency in adult neural stem cells defines a pathological protein-network signature and defective neurogenesis in a mouse model of AD.

doi: 10.1186/s13287-024-03805-1

Figure Lengend Snippet: Fig. 2 Nup153 overexpression in AD-NSCs counteracts oxidative stress and recovers proteasomal activity. (A) Immunofluorescence showing GFP expres sion in AD-NSCs transfected with GFP or GFP-Nup153 vectors at 72 h. (B) DHE labelling of NSCs in the above conditions to reveal the level of oxidative stress. The graph indicates the mean fluorescence intensity for DHE (n = 4). (C) Proteasomal activity measured in WT-, AD-GFP- and AD-GFP-Nup153-NSCs (n = 5). *P < 0.05. Scale bar 20 μm

Article Snippet: All mice were randomly assigned to receive Nup153 or GFP-control lentiviral particles (1.5 μl; 1 × 109/ml Origene CW305175V: lentiviral particles produced from PS100071 and MR211997L4) that were delivered in the hippocampus by a stereotaxic injection.

Techniques: Over Expression, Activity Assay, Immunofluorescence, Transfection, Fluorescence

Journal: Stem cell research & therapy

Article Title: Nucleoporin 153 deficiency in adult neural stem cells defines a pathological protein-network signature and defective neurogenesis in a mouse model of AD.

doi: 10.1186/s13287-024-03805-1

Figure Lengend Snippet: Fig. 6 Nup153 transduction in AD-iPSCs improves the differentiation of brain cortical organoids. (A) Western blot analysis of MAP2, β3 tubulin, DCX, NeuN and NF proteins in brain organoids (n = 3) and relative quantification based on the expression of actin. Each lysate was obtained from the pool of 3–4 individual organoids. (B, D) Representative images of syn I and PSD95 expression from control, AD and AD-Nup153 brain organoids analyzed by confocal analysis and counterstained with MAP2 and DAPI (scale bar 50 μm) at 2 months of differentiation. (C, E) Quantification of mean fluorescence intensity of SynI and PSD95 puncta/area relative to control, AD and AD-Nup153 brain organoids (n = 3). *P < 0.05

Article Snippet: All mice were randomly assigned to receive Nup153 or GFP-control lentiviral particles (1.5 μl; 1 × 109/ml Origene CW305175V: lentiviral particles produced from PS100071 and MR211997L4) that were delivered in the hippocampus by a stereotaxic injection.

Techniques: Transduction, Western Blot, Quantitative Proteomics, Expressing, Control, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: A role for Nup153 in nuclear assembly reveals differential requirements for targeting of nuclear envelope constituents

doi: 10.1091/mbc.e22-05-0189

Figure Lengend Snippet: FIGURE 1: Lamins B1/B2 accumulate at ectopic sites in telophase when Nup153 is depleted. (A) Confocal microscopy of HeLa cells expressing LEM2-mCherry, costained to detect Nup153, tubulin, and DNA. Images represent distinct stages of nuclear assembly, from initial membrane recruitment in early anaphase, establishment of core/noncore regions in late anaphase, and dissolution of core/noncore regions along with nuclear envelope expansion (early telophase, late telophase). Early telophase cells are identified by a compact midbody structure connecting cells that have not fully flattened, while late-telophase cells remain connected by a thin midbody structure and are considerably flatter. Scale bar, 10 µm. (B) Widefield microscopy showing representative images of lamins A/C, B1, and B2 in late-telophase stage cells, after treatment with siControl (siCon) or siNup153 (si153) oligos for 48 h. Yellow arrows indicate sites of lamin ectopic targeting. Scale bar, 20 µm. (C) Quantification of the percent of cells with mislocalized endogenous lamin B2 at metaphase/anaphase (Meta/Ana), early telophase (Early Telo), and late telophase (Late Telo) after 48-h treatment with siCon or si153 oligos. The phenotype observed in early telophase cells was notably weaker. Data from three experiments plotted as mean ± SD; siCon: meta/ana 0 ± 0%, early telo 0 ± 0%, late telo 0 ± 0%; si153: meta/ana 0 ± 0%, early telo 38 ± 15%, late telo 80 ± 13%. *, P < 0.05; ***, P < 0.001. Ø indicates raw values were equal to 0. (D) Montage of cells expressing GFP-lamin B2 and H2B-mCherry progressing through telophase captured by live spinning disk microscopy after treatment with siCon or si153 for 48 h, with t = 0′ being the time of complete cleavage furrow ingression observed by time-lapse video. Images were adjusted independently to optimize visibility of the markers. Scale bar, 10 µm.

Article Snippet: Primary antibodies are as follows: rabbit α-aurora B pT232 (Rockland; 600-401-677); mouse α-lamin A/C (Cell Signaling; 4C11); rabbit α-lamin B1 (Abcam; ab16408); mouse α-lamin B2 (Abcam; ab8983); hamster α-Lap2α (gift from Joe Glavy, University of Texas at Tyler); mouse α-Lap2β (gift from Brian Burke, Skin Research Institute of Singapore); rabbit α-LBR (Abcam; ab32535); rabbit α-Nup153 (Bethyl; 301-788A); mouse α-Nup153 (SA1; gift from Brian Burke); rabbit α-POM121 (GeneTex; GTX102128); rabbit α-SUN1 (Abcam; ab124770); mouse α-SUN2 (gift from Brian Burke); rat α-RFP (Proteintech; 5f8); rabbit α-tubulin (Abcam; ab18251); rat α-tubulin (Abcam; ab6160); chicken α-tubulin (Synaptic Systems; 302 206); sheep α-tubulin (Cytoskeleton; ATN02); followed by AlexaFluor conjugated secondary antibodies (ThermoFisher).

Techniques: Confocal Microscopy, Expressing, Membrane, Dissolution, Microscopy

Journal: Molecular Biology of the Cell

Article Title: A role for Nup153 in nuclear assembly reveals differential requirements for targeting of nuclear envelope constituents

doi: 10.1091/mbc.e22-05-0189

Figure Lengend Snippet: FIGURE 2: Lamin B2 mistargeting is not the result of disrupting the pore basket. (A) Immunoblot of Nup153, Nup50, Tpr, Nup155, and tubulin protein levels after depletion with indicated siRNAs for 48 h. Nup155 and tubulin were probed as nucleoporin and loading controls, respectively. (B) Quantification of the percent of late-telophase cells with mistargeted lamin B2 after treatment with the indicated siRNAs. Data from three experiments plotted as mean ± SD; siCon: 0 ± 0%; si153: 90 ± 10%; si50: 0 ± 0%; siTpr: 0 ± 0%. ***, P < 0.001. Ø indicates raw values were equal to 0. (C) Widefield microscopy of lamin B2 for each depletion condition; brackets indicate late- telophase cells determined by tubulin costain (not shown). Scale bar, 20 µm.

Article Snippet: Primary antibodies are as follows: rabbit α-aurora B pT232 (Rockland; 600-401-677); mouse α-lamin A/C (Cell Signaling; 4C11); rabbit α-lamin B1 (Abcam; ab16408); mouse α-lamin B2 (Abcam; ab8983); hamster α-Lap2α (gift from Joe Glavy, University of Texas at Tyler); mouse α-Lap2β (gift from Brian Burke, Skin Research Institute of Singapore); rabbit α-LBR (Abcam; ab32535); rabbit α-Nup153 (Bethyl; 301-788A); mouse α-Nup153 (SA1; gift from Brian Burke); rabbit α-POM121 (GeneTex; GTX102128); rabbit α-SUN1 (Abcam; ab124770); mouse α-SUN2 (gift from Brian Burke); rat α-RFP (Proteintech; 5f8); rabbit α-tubulin (Abcam; ab18251); rat α-tubulin (Abcam; ab6160); chicken α-tubulin (Synaptic Systems; 302 206); sheep α-tubulin (Cytoskeleton; ATN02); followed by AlexaFluor conjugated secondary antibodies (ThermoFisher).

Techniques: Western Blot, Microscopy

Journal: Molecular Biology of the Cell

Article Title: A role for Nup153 in nuclear assembly reveals differential requirements for targeting of nuclear envelope constituents

doi: 10.1091/mbc.e22-05-0189

Figure Lengend Snippet: FIGURE 3: SUN1, but not SUN2, enriches at ectopic sites in late-telophase cells when Nup153 is depleted. (A) Widefield microscopy of SUN1 and tubulin and SUN2 and tubulin (B) in late-telophase cells following treatment with siCon or si153 under thymidine-synchronization conditions. Scale bar, 20 µm. (C) Quantification of the percent late- telophase cells with ectopic SUN1 targeting. Data from three experiments plotted as mean ± SD; siCon: 1 ± 1%; si153: 73 ± 13%; ***, P < 0.001. (D) Relative levels of SUN1 and SUN2 at ectopic foci compared with nuclear levels under si153 conditions. Data collected from three independent biological replicates are depicted in different colors, N = 25, 26, 20; the mean of each experiment was used to calculate the mean ± SD, indicated in black; SUN1: 2.0 ± 0.3%; SUN2: 0.5 ± 0.1%. (E) Graphed arbitrary fluorescence of SUN1 and SUN2 along plot line profile indicated on the spinning disk confocal micrographs shown in F. Scale bar in F, 10 µm.

Article Snippet: Primary antibodies are as follows: rabbit α-aurora B pT232 (Rockland; 600-401-677); mouse α-lamin A/C (Cell Signaling; 4C11); rabbit α-lamin B1 (Abcam; ab16408); mouse α-lamin B2 (Abcam; ab8983); hamster α-Lap2α (gift from Joe Glavy, University of Texas at Tyler); mouse α-Lap2β (gift from Brian Burke, Skin Research Institute of Singapore); rabbit α-LBR (Abcam; ab32535); rabbit α-Nup153 (Bethyl; 301-788A); mouse α-Nup153 (SA1; gift from Brian Burke); rabbit α-POM121 (GeneTex; GTX102128); rabbit α-SUN1 (Abcam; ab124770); mouse α-SUN2 (gift from Brian Burke); rat α-RFP (Proteintech; 5f8); rabbit α-tubulin (Abcam; ab18251); rat α-tubulin (Abcam; ab6160); chicken α-tubulin (Synaptic Systems; 302 206); sheep α-tubulin (Cytoskeleton; ATN02); followed by AlexaFluor conjugated secondary antibodies (ThermoFisher).

Techniques: Microscopy, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: A role for Nup153 in nuclear assembly reveals differential requirements for targeting of nuclear envelope constituents

doi: 10.1091/mbc.e22-05-0189

Figure Lengend Snippet: FIGURE 4: Mistargeted nuclear envelope proteins coenrich at ectopic sites that are membranous but distinct from micronuclei. (A) Representative widefield images of lamin B1 and B2 in Nup153-depleted cells after thymidine synchronization. Enlargements are 2×. Scale bar, 20 µm. (B) Spinning disk confocal micrographs showing lamin B2 and SUN1 after Nup153 depletion. Scale bar, 20 µm. (C) Arbitrary fluorescence units corresponding to the plot line shown in B are graphed. (D) Spinning disk confocal micrographs showing Lap2β and SUN1 after Nup153 depletion. Scale bar, 20 µm. (E) Arbitrary fluorescence units corresponding to the plot line shown in D are graphed. (F) Costain of SUN1 and Lap2α in Nup153-depleted cells by widefield microscopy, distinguish micronucleus from sites of SUN1 accumulation. Scale bar, 20 µm. (G) Live spinning disk confocal microscopy of BFP-Sec61β and GFP-lamin B2 in si153- treated cells, showing continuity of ER membrane with ectopic site of lamin B2 localization. Enlargements are 2.5×. Scale bar, 10 µm.

Article Snippet: Primary antibodies are as follows: rabbit α-aurora B pT232 (Rockland; 600-401-677); mouse α-lamin A/C (Cell Signaling; 4C11); rabbit α-lamin B1 (Abcam; ab16408); mouse α-lamin B2 (Abcam; ab8983); hamster α-Lap2α (gift from Joe Glavy, University of Texas at Tyler); mouse α-Lap2β (gift from Brian Burke, Skin Research Institute of Singapore); rabbit α-LBR (Abcam; ab32535); rabbit α-Nup153 (Bethyl; 301-788A); mouse α-Nup153 (SA1; gift from Brian Burke); rabbit α-POM121 (GeneTex; GTX102128); rabbit α-SUN1 (Abcam; ab124770); mouse α-SUN2 (gift from Brian Burke); rat α-RFP (Proteintech; 5f8); rabbit α-tubulin (Abcam; ab18251); rat α-tubulin (Abcam; ab6160); chicken α-tubulin (Synaptic Systems; 302 206); sheep α-tubulin (Cytoskeleton; ATN02); followed by AlexaFluor conjugated secondary antibodies (ThermoFisher).

Techniques: Fluorescence, Microscopy, Confocal Microscopy, Membrane

Journal: Molecular Biology of the Cell

Article Title: A role for Nup153 in nuclear assembly reveals differential requirements for targeting of nuclear envelope constituents

doi: 10.1091/mbc.e22-05-0189

Figure Lengend Snippet: FIGURE 5: LBR accumulates ectopically in late-telophase stage cells depleted of Nup153. (A) Widefield images of LBR and lamin B2 detection in late-telophase cells, determined by tubulin staining (unpublished data), following treatment with siCon and si153 oligos. Scale bar, 20 μm. Enlargements are 2X. (B) Quantification of cytoplasmic to nuclear ratio for LBR. Data collected from three biological replicates are depicted in different colors, N = 71, 68, 36 (siCon) and 72, 69, 36 (si153). The mean of each experiment was used to calculate the mean ± SD, indicated in black; siCon: 0.39 ± 0.03; si153: 0.67 ± 0.04. (C) Confocal image of LBR and lamin B2 codetection in siNup153-treated cells. Scale bar, 20 µm. (D) Arbitrary fluorescence units corresponding to the plot line shown in C are graphed.

Article Snippet: Primary antibodies are as follows: rabbit α-aurora B pT232 (Rockland; 600-401-677); mouse α-lamin A/C (Cell Signaling; 4C11); rabbit α-lamin B1 (Abcam; ab16408); mouse α-lamin B2 (Abcam; ab8983); hamster α-Lap2α (gift from Joe Glavy, University of Texas at Tyler); mouse α-Lap2β (gift from Brian Burke, Skin Research Institute of Singapore); rabbit α-LBR (Abcam; ab32535); rabbit α-Nup153 (Bethyl; 301-788A); mouse α-Nup153 (SA1; gift from Brian Burke); rabbit α-POM121 (GeneTex; GTX102128); rabbit α-SUN1 (Abcam; ab124770); mouse α-SUN2 (gift from Brian Burke); rat α-RFP (Proteintech; 5f8); rabbit α-tubulin (Abcam; ab18251); rat α-tubulin (Abcam; ab6160); chicken α-tubulin (Synaptic Systems; 302 206); sheep α-tubulin (Cytoskeleton; ATN02); followed by AlexaFluor conjugated secondary antibodies (ThermoFisher).

Techniques: Staining, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: A role for Nup153 in nuclear assembly reveals differential requirements for targeting of nuclear envelope constituents

doi: 10.1091/mbc.e22-05-0189

Figure Lengend Snippet: FIGURE 6: Nup153 plays a critical role specific to NE formation. (A) Immunoblot of DLD1- Nup153AID cells growing asynchronously or in the presence of thymidine to prevent progression into mitosis. Cells were treated with auxin for 15 h, as indicated. Following detection of Nup153 and lamin B2, blots were reprobed for vinculin as a loading control. (B) Widefield microscopy of cells treated as in A tracking the appearance of endogenous lamin B2 with DNA counterstained. Images were adjusted independently to best illustrate lamin B2 distribution pattern. Enlargements are 2×. Scale bar, 20 μM. (C) Quantification of nuclei with associated lamin B2 foci. Data collected from three biological replicates are graphed using the mean from each experiment to calculate the overall mean ± SD; no thymidine, no auxin 8.5 ± 0.9% (N = 1078, 771, 784); no thymidine, plus auxin 53.6 ± 5.4% (N = 664, 538, 465); thymidine, no auxin 1.6 ± 0.4% (N = 486, 404, 389); thymidine, plus auxin 6.8 ± 2.5% (N = 460, 353, 509). *, P < 0.05; ***, P < 0.001.

Article Snippet: Primary antibodies are as follows: rabbit α-aurora B pT232 (Rockland; 600-401-677); mouse α-lamin A/C (Cell Signaling; 4C11); rabbit α-lamin B1 (Abcam; ab16408); mouse α-lamin B2 (Abcam; ab8983); hamster α-Lap2α (gift from Joe Glavy, University of Texas at Tyler); mouse α-Lap2β (gift from Brian Burke, Skin Research Institute of Singapore); rabbit α-LBR (Abcam; ab32535); rabbit α-Nup153 (Bethyl; 301-788A); mouse α-Nup153 (SA1; gift from Brian Burke); rabbit α-POM121 (GeneTex; GTX102128); rabbit α-SUN1 (Abcam; ab124770); mouse α-SUN2 (gift from Brian Burke); rat α-RFP (Proteintech; 5f8); rabbit α-tubulin (Abcam; ab18251); rat α-tubulin (Abcam; ab6160); chicken α-tubulin (Synaptic Systems; 302 206); sheep α-tubulin (Cytoskeleton; ATN02); followed by AlexaFluor conjugated secondary antibodies (ThermoFisher).

Techniques: Western Blot, Control, Microscopy

Journal: Molecular Biology of the Cell

Article Title: A role for Nup153 in nuclear assembly reveals differential requirements for targeting of nuclear envelope constituents

doi: 10.1091/mbc.e22-05-0189

Figure Lengend Snippet: FIGURE 7: Summary model of observations. After the genome segregates in early anaphase, nuclear envelope proteins dispersed throughout the mitotic ER seed the formation of the nascent nuclear envelope at the chromatin surface. By late anaphase, INM proteins form distinct core and noncore domains at NE. As cells exit late anaphase and enter telophase, these transient domains redistribute as midzone microtubules condense into a midbody structure. During the progression from late anaphase to telophase, Nup153 is required for the continued targeting of primarily noncore INM proteins. At interphase, INM protein targeting occurs independently of Nup153 with the exception to-date of LBR.

Article Snippet: Primary antibodies are as follows: rabbit α-aurora B pT232 (Rockland; 600-401-677); mouse α-lamin A/C (Cell Signaling; 4C11); rabbit α-lamin B1 (Abcam; ab16408); mouse α-lamin B2 (Abcam; ab8983); hamster α-Lap2α (gift from Joe Glavy, University of Texas at Tyler); mouse α-Lap2β (gift from Brian Burke, Skin Research Institute of Singapore); rabbit α-LBR (Abcam; ab32535); rabbit α-Nup153 (Bethyl; 301-788A); mouse α-Nup153 (SA1; gift from Brian Burke); rabbit α-POM121 (GeneTex; GTX102128); rabbit α-SUN1 (Abcam; ab124770); mouse α-SUN2 (gift from Brian Burke); rat α-RFP (Proteintech; 5f8); rabbit α-tubulin (Abcam; ab18251); rat α-tubulin (Abcam; ab6160); chicken α-tubulin (Synaptic Systems; 302 206); sheep α-tubulin (Cytoskeleton; ATN02); followed by AlexaFluor conjugated secondary antibodies (ThermoFisher).

Techniques:

Journal: Nature Communications

Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding

doi: 10.1038/s41467-020-16394-3

Figure Lengend Snippet: a Silver stain showing proteins that immunoprecipitated (IP) with FLAG-NUP153. b Co-IP shows FLAG-NUP153 interaction with CTCF, and cohesin subunits, SMC3, SMC1A, and RAD21. NUP153 was pulled down using anti-FLAG antibody. c Schematic showing steps of chromatin fractionation assay in HeLa cells. d NUP153 was detected in the nuclear insoluble fraction (P2) along with CTCF and cohesin. NUP153 detected in the chromatin-associated soluble fraction (S3) following micrococcal nuclease (MNase) treatment of P1 fraction. Ppt, precipitate; Sup, supernatant; Nucleoporin 62, NUP62; loading controls: α-TUBULIN (cytoplasm), Histone H3 (chromatin). Experiments were repeated twice. Source data are provided as a file.

Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full-length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP153 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

Techniques: Silver Staining, Immunoprecipitation, Co-Immunoprecipitation Assay, Fractionation

Journal: Nature Communications

Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding

doi: 10.1038/s41467-020-16394-3

Figure Lengend Snippet: a Distribution of NUP153 peaks in mouse ES cells. Peaks are categorized as promoters (−2 kb from TSS to +100 bp from TSS), gene body (+100 bp from TSS to +1 kb from transcription termination site (TTS)), intergenic sites (<−2 kb from TSS and >+1 kb from TTS). See Supplementary Data for a list of NUP153 binding sites. b Metagene profiles of mean NUP153 binding at NUP153-positive and NUP153-negative TSS and enhancer (±5 kb), and TAD boundaries (±250 kb) (top). Number and percentage of NUP153 binding sites are presented as a table for the indicated genetic elements (bottom). (See Supplementary Data for NUP153 binding sites at different genetic elements.) c Genome-wide CTCF and SMC3 binding sites were compared in control and NUP153 deficient (KD-1, KD-2) mouse ES cells. d Metagene profiles showing mean CTCF and SMC3 binding across CTCF-positive TSS ( n = 2164), enhancers ( n = 2272), and TAD boundaries ( n = 2238) in control and NUP153 deficient mouse ES cells. e Mean CTCF binding in control and NUP153 KD mouse ES cells were compared and CTCF sites were grouped into two. Group I contained CTCF sites that showed greater mean CTCF binding in control cells over NUP153 KD cells. Group II contained CTCF sites that showed equal or lesser mean CTCF binding in control cells over NUP153 KD cells. Number of CTCF-positive sites across TSS and enhancer (±2.5 kb) and TAD boundaries (±250 kb), and the number of NUP153 target genes that associate with each group are shown as a table (top) (see also Supplementary Data and Supplementary Fig. ). Metagene profiles showing mean NUP153 binding across CTCF-positive Group I and Group II TSS, enhancer (±2.5 kb) and TAD boundaries (±250 kb) (bottom).

Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full-length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP153 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

Techniques: Binding Assay, Genome Wide, Control

Journal: Nature Communications

Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding

doi: 10.1038/s41467-020-16394-3

Figure Lengend Snippet: a Scatter plot showing expression levels of transcripts in log2[CPM] scale in control and NUP153 KD-1 mouse ES cells. Blue points denote all differentially expressed genes ( n = 711) and orange points denote differentially expressed bivalent genes (Supplementary Data and ). b Table showing number of differentially expressed genes that associate with all, Group I and Group II CTCF-positive TSS (top). Plots showing number of differentially regulated NUP153-positive and NUP153-negative genes that associate with Group I and II CTCF-positive TSS (bottom) (see also Supplementary Data ). c NUP153 DamID-Seq, CTCF, cohesin, H3K4me3, and H3K27me3 ChIP-Seq, and RNA-Seq tracks are shown for two NUP153-positive Group I genes, Rtn4rl1 (left panel) and Calb2 (right panel) in control (WT) and NUP153 KD ES cells. Rtn4rl1 shows transcriptional upregulation and Calb2 shown transcriptional downregulation. d NUP153 DamID-Seq, CTCF, cohesin, H3K4me3, and H3K27me3 ChIP-Seq tracks are shown for a 145–150 kb region for the HoxA and HoxC loci in control (WT) and NUP153 KD mouse ES cells as indicated. Arrows point to regions where CTCF or SMC3 binding are altered in NUP153 KD mouse ES cells. CTCF sites labeled with asterisk (*) denote CTCF sites that have been reported to regulate transcription at the Hox loci by mediating the formation of TADs , . The 2D heat map shows the interaction frequency in mouse ES cells . Hi-C data was aligned to the mm9 genome showing HoxA cluster residing in a TAD boundary and HoxC cluster in a TAD as published . H3K4me3 and H3K27me3 (ref. ) and CBP/P300 (ref. ) ChIP-Seq data were previously published. CPM, counts per million.

Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full-length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP153 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

Techniques: Expressing, Control, ChIP-sequencing, RNA Sequencing, Binding Assay, Labeling, Hi-C

Journal: Nature Communications

Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding

doi: 10.1038/s41467-020-16394-3

Figure Lengend Snippet: a Western blot showing NUP153 protein levels in control and NUP153 KD HeLa cells. b Real-time RT-PCR showing relative IEG mRNA and nascent mRNA levels in control and NUP153 KD HeLa cells in a time course dependent manner. JUN mRNA levels at minus EGF (control vs KD-1, ** p = 0.0033; control vs KD-2, * p = 0.0345), 15 min EGF (control vs KD-1, * p = 0.0138; control vs KD-2, * p = 0.0199), 30 min EGF (control vs KD-1, *** p = 0.0001; control vs KD-2, *** p = 0.0001), 60 min EGF (control vs KD-1, ** p = 0.0010; control vs KD-2, *** p = 0.0004), 90 min EGF (control vs KD-1, ** p = 0.0065; control vs KD-2, ** p = 0.0045), 120 min EGF (control vs KD-1, ** p = 0.0010; control vs KD-2, ** p = 0.0050). EGR1 mRNA levels at minus EGF (control vs KD-1, ** p = 0.0018; control vs KD-2, *** p = 0.0008), 15 min EGF (control vs KD-1, * p = 0.0373; control vs KD-2, ** p = 0.0066). EGR1 pre-mRNA levels at 15 min EGF (control vs KD-1, ** p = 0.0038; control vs KD-2, * p = 0.0151), 30 min EGF (control vs KD-1, ** p = 0.0045; control vs KD-2, * p = 0.0436), 90 min EGF (control vs KD-1, * p = 0.0364; control vs KD-2, * p = 0.0305). c Real-time RT-PCR showing relative JUN and NUP153 mRNA levels in control, NUP153 KD and FLAG-NUP153 expressing NUP153 KD HeLa cells upon 15 min EGF treatment. GAPDH was used to normalize mRNA levels. JUN mRNA levels in control vs KD (*** p = 0.0000); KD vs FLAG-NUP153 (*** p = 0.0001). NUP153 mRNA levels in control vs KD (** p = 0.0011); KD vs FLAG-NUP153 (*** p = 0.0000). d POL II binding across JUN and EGR1 genetic elements was mapped by ChIP real-time PCR in control and NUP153 KD HeLa cells under indicated conditions (see Supplementary Data for primer sequences). POL II binding at JUN , minus EGF (control vs KD; promoter, * p = 0.0329; TSS, ** p = 0.0042; GB, *** p = 0.0003; TTS, * p = 0.0493), 15 min EGF (control vs KD; promoter, *** p = 0.0009; TSS, ** p = 0.0065; GB, *** p = 0.0009; TTS, * p = 0.0443), 30 min EGF (control vs KD; GB, * p = 0.0385). POL II binding at EGR1 , minus EGF (control vs KD; promoter, * p = 0.0172; TSS, ** p = 0.0060), 15 min EGF (control vs KD; TSS, *** p = 0.0004; TTS, * p = 0.0281). Data shown are percent (%) of input. Values are mean ± standard deviation. Two-tailed Student’s t -test, n ≥ 3 independent experiments. Nt, nucleotide. Source data are provided as a file.

Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full-length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP153 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

Techniques: Western Blot, Control, Quantitative RT-PCR, Expressing, Binding Assay, Real-time Polymerase Chain Reaction, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding

doi: 10.1038/s41467-020-16394-3

Figure Lengend Snippet: NUP153, CTCF, and SMC3 occupancy across the JUN (left) and EGR1 (right) genetic elements were examined by ChIP real-time PCR at the indicated time points in control and NUP153 KD HeLa cells. Position of PCR primers are denoted as numbers in the schematics (see Supplementary Data for primer sequences). NUP153 binding at JUN , minus EGF (control vs KD; TSS, *** p = 0.0000; GB, ** p = 0.0035; TTS, ** p = 0.0011; +14.6 kb, ** p = 0.0023), 15 min EGF (control vs KD; promoter, ** p = 0.0047; TSS, * p = 0.0368; GB, *** p = 0.0003; TTS, *** p = 0.0004; +14.6 kb, * p = 0.0301). NUP153 binding at EGR1 , minus EGF (control vs KD; −15.3 kb, * p = 0.0298; −1.2 kb, * p = 0.0323; −0.9 kb, ** p = 0.0039; promoter, * p = 0.0469; TTS, * p = 0.0406), 15 min EGF (control vs KD; −15.5 kb, * p = 0.0299; −15.3 kb, ** p = 0.0068; −1.2 kb, *** p = 0.0006; −0.9 kb, ** p = 0.0086; promoter, *** p = 0.0006; TSS, ** p = 0.0078; TTS, ** p = 0.0085). CTCF binding at JUN , minus EGF (control vs KD; GB, ** p = 0.0047; +14.6 kb, * p = 0.0231). CTCF binding at EGR1 , minus EGF (control vs KD; −15.3 kb, ** p = 0.0017; −1.2 kb, ** p = 0.0010; −0.9 kb, * p = 0.0291), 15 min EGF (control vs KD; −15.3 kb, * p = 0.0316). SMC3 binding at JUN , minus EGF (control vs KD; −1.3 kb, ** p = 0.0076; promoter, ** p = 0.0032; TSS, ** p = 0.0037; GB, * p = 0.0413; +14.6 kb, * p = 0.0158), 15 min EGF (control vs KD; −1 kb, * p = 0.0154; TSS, ** p = 0.0029; GB, ** p = 0.0036; +14.6 kb, * p = 0.0245). SMC3 binding at EGR1 , minus EGF (control vs KD; −15.5 kb, *** p = 0.0001; −15.3 kb, * p = 0.0309), 15 min EGF (control vs KD; −15.3 kb, *** p = 0.0004; −1.2 kb, * p = 0.0355; −0.9 kb, * p = 0.0419; TTS, *** p = 0.0003). Data shown are percent (%) of input. Values are mean ± standard deviation. Two-tailed Student’s t -test, n ≥ 3 independent experiments. Nt, nucleotide. Source data are provided as a file.

Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full-length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP153 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

Techniques: Real-time Polymerase Chain Reaction, Control, Binding Assay, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding

doi: 10.1038/s41467-020-16394-3

Figure Lengend Snippet: a Real-time RT-PCR showing relative CTCF mRNA levels in HeLa cells transfected with control or CTCF shRNA expression vectors (KD-1 and KD-2). Control vs KD-1, *** p = 0.0000; control vs KD-2, *** p = 0.0000. b Real-time RT-PCR showing relative JUN and EGR1 mRNA levels in control or CTCF KD HeLa cells. JUN mRNA levels at minus EGF (control vs KD-1, * p = 0.0126; control vs KD-2, ** p = 0.0011), 15 min EGF (control vs KD-1, ** p = 0.0077; control vs KD-2, ** p = 0.0011), 30 min EGF (control vs KD-1, *** p = 0.0000; control vs KD-2, *** p = 0.0000), 120 min EGF (control vs KD-2, * p = 0.0299). EGR1 mRNA levels at minus EGF (control vs KD-2, * p = 0.0473), 15 min EGF (control vs KD-1, ** p = 0.0012; control vs KD-2, ** p = 0.0050), 30 min EGF (control vs KD-1, * p = 0.0162; control vs KD-2, *** p = 0.0000). c POL II binding across the IEGs, JUN and EGR1 loci, was mapped by POL II ChIP in control and CTCF KD HeLa cells at the paused state (minus EGF). POL II occupancy at the indicated genetic elements was measured using primers as denoted in the schematics (Supplementary Data ). POL II binding at JUN promoter (control vs KD-1; * p = 0.0261), TSS ((control vs KD-1; ** p = 0.0012), (control vs KD-2; ** p = 0.0087)), GB (control vs KD-2; * p = 0.0463); EGR1 promoter ((control vs KD-1; * p = 0.0209), (control vs KD-2; ** p = 0.0038)), TSS ((control vs KD-1; ** p = 0.0048), (control vs KD-2; ** p = 0.0011)). Data shown are percent (%) of input. d Real-time RT-PCR showing relative EGR1, JUN, NUP153, and CTCF mRNA levels in NUP153 KD, CTCF KD, and in CTCF/NUP153 KD HeLa cells. JUN mRNA levels (control vs NUP153 KD, *** p = 0.0009; control vs CTCF-KD, *** p = 0.0006; control vs NUP153 and CTCF KD, *** p = 0.0002). EGR1 mRNA levels (control vs NUP153 KD, *** p = 0.0003; control vs CTCF-KD, *** p = 0.0000; control vs NUP153 and CTCF KD, *** p = 0.0001). NUP153 mRNA levels (control vs NUP153 KD, *** p = 0.0000; control vs NUP153 and CTCF KD, *** p = 0.0000). CTCF mRNA levels (control vs CTCF-KD, *** p = 0.0000; control vs NUP153 and CTCF KD, *** p = 0.0005). Values are mean ± standard deviation. Relative mRNA levels were normalized using GAPDH. Two-tailed Student’s t -test, n ≥ 3 independent experiments. NS, not significant ( p > 0.05). Nt, nucleotide. Source data are provided as a file.

Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full-length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP153 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

Techniques: Quantitative RT-PCR, Transfection, Control, shRNA, Expressing, Binding Assay, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding

doi: 10.1038/s41467-020-16394-3

Figure Lengend Snippet: a Immunostaining of LAMIN B1 and c-FOS DNA FISH in control and NUP153 KD HeLa cells are shown at the indicated time points. Cell numbers are as indicated. HeLa cells contain three c-FOS alleles (white arrows). Scale bar, 5 μm. b Cumulative frequency graphs showing distribution of the c-FOS locus distance to nuclear periphery in control and NUP153 KD HeLa cells at the indicated time points. Cumulative frequencies at a normalized distance (ND) of 0.0–0.12 are shown. See Supplementary Fig. for the distribution of loci in all cells that were analyzed (ND of 0.0–0.5). ND = c-FOS locus to periphery distance/cell diameter ( d ), where d = (2× nuclear area/ π ) 0.5 . * p < 0.05; *** p < 0.001; Kolmogorov–Smirnov (KS)-test, n = 2 independent experiments. c Working model showing NUP153-mediated chromatin structure and transcription regulation at the IEG locus. Source data are provided as a file.

Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full-length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP153 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

Techniques: Immunostaining, Control