Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Nuclear pore protein TPR associates with lamin B1 and affects nuclear lamina organization and nuclear pore distribution

doi: 10.1007/s00018-019-03037-0

Figure Lengend Snippet: A distribution of NPCs is affected in TPR-depleted HeLa cells (shTPR HeLa cell line). a–c The distribution of NPCs in control HeLa cells as viewed by immunostaining with anti-Nup62 antibody and wide-field Leica DM6000 microscope. a Homogenous distribution. b Small NPC-free zones. c Large NPC-free zones comprehend more than 1/10 of nuclear surface. d The ratio of homogenous distribution, small and large NPC-free zones in control wild-type S-G2 cells (wt), in cells stably expressing shRNA targeting non-coding regions (shNC) and TPR (shTPR cells). In TPR-depleted cells, the number of nuclei with NPC-free zones increased significantly (χ2 = 11.41, df = 2, P < 0.001, Chi squared test), **P < 0.01, ***P < 0.001, n.s. not significant. e The ratio of homogenous, small and large NPC-free zones in control wild type cells (wt) and in cells stably expressing shRNA targeting non-coding regions (shNC) and TPR (shTPR) synchronized in G1/S and in G2 phase of the cell cycle. The number of NPC-free zones significantly increased in TPR-depleted cells (***P < 0.001, n.s. not significant). f–i Recruitment of nucleoporins Nup107, Nup133 and central FG-Nups into NPC-free zones is impaired in TPR-depleted cells. f The ratio of distribution of NPC-free zones in shNC and shTPR cells stained with Nup153 antibody (χ2 = 7.08, df = 2, P = 0.029). g The ratio of homogenous distribution, small and large NPC-free zones in shNC and shTPR cells transfected with GFP–Nup107 construct (χ2 = 9.713, df = 2, P = 0.008). h The ratio of homogenous distribution, small and large NPC-free zones in shNC and shTPR cells overexpressing GFP–Nup133 (χ2 = 23.855, df = 2, P < 0.001). i The ratio of distribution of NPC-free zones in shNC and shTPR cells stained with antibody recognizing central FG-Nups (χ2 = 138.5, df = 2, P < 0.0001)

Article Snippet: Antibodies Primary antibodies were used as follows:. anti-TPR mouse monoclonal (nTPR, ab58344, Abcam, Cambridge, UK, 1:300), anti-TPR rabbit polyclonal (cTPR, ab84516, Abcam, Cambridge, UK, 1:300), anti-lamin B1 rabbit polyclonal IgG (ab16048, Abcam, Cambridge, UK, 1:100), anti-lamin A/C mouse monoclonal (SAB4200236, Sigma, Sigma-Aldrich, St. Louis, USA, 1:100), anti-Nup 62 rat monoclonal IgG (ab188413, Abcam, Cambridge, UK, 1:500); anti-Nup153 rabbit polyclonal antibody (NB100-93329, Novus Biologicals, 1:200), Nuclear pore complex proteins (ab24609, Abcam Cambridge, UK, 1:200), anti-GFP mouse monoclonal (ab1218, Abcam, Cambridge, UK, 1:200, anti-GFP rabbit polyclonal (1828014, Molecular probes at Thermofisher Scientific, Waltham, MA USA, 1:200), anti-PCNA human polyclonal, anti-tubulin α (N-terminal structural domain, TU-01, aa 65–79, 1:100) mouse monoclonal was kindly provided by Dr. Pavel Draber (Institute of Molecular Genetics of the ASCR, v.v.i., Prague, Czech Republic).

Techniques: Control, Immunostaining, Microscopy, Stable Transfection, Expressing, shRNA, Staining, Transfection, Construct

Journal: Cardiovascular research

Article Title: The nuclear pore protein Nup153 associates with chromatin and regulates cardiac gene expression in dystrophic mdx hearts.

doi: 10.1093/cvr/cvw204

Figure Lengend Snippet: Figure 1 Nup153 protein expression is up-regulated in the mdx heart and controlled by NO availability. (A) Representative images of Nup153 (red) expression in WT and mdx ventricles counterstained with a-sarcomeric actin (green) or with DAPI (scale bar: 20lm). 3D plots relative to Nup153 expres- sion are shown in (B). (C and D) Mean fluorescence intensities (3.56 0.59WT vs. 22.96 6.8 mdx mice) and the percentage of Nup153þnuclei (22.46 7.8% WT vs. 56.46 12.5% mdx mice) in ventricle sections (n¼ 4 control, n ¼ 5 mdx mice, *P < 0.01, t-test). (E) Nup153 level in WT and mdx mice by WB (two representative animals per group are shown) and densitometry (mean fold increase), *P < 0.05 vs. WT, Mann–Whitney. (F and G) Nup153 protein level in WT and mdx heart organotypic cultures treated or not with DETANO (100, 500 lM for 24h). The graphs show the densitometry (*P < 0.01 vs. WT,** P < 0.05 vs. mdx, §P < 0.01 vs. mdx, ANOVA followed by SNK post hoc, n ¼ 5). (H) Representative confocal images showing Nup153 (red) in Hl-1 cells untreated (C) or treated with LNAME (LN, 5 mM, 24h). Nuclei were counterstained with DAPI (scale bar: 50lm, n ¼ 4). Right panel shows the mean fluorescence intensities relative to control and LNAME-treated cells (19.816 3.8 and 40.436 5.8, respectively, *P < 0.05 vs. control, t-test. (I) Nup153 pro- tein level in HL-1 cells treated with LNAME (0.1–1–5–10 mM) for 24h (n ¼ 3). Lower panel shows the densitometric analysis (*P < 0.05 vs. control, Kruskal–Wallis on ranks followed by Dunnet post hoc). (J) Nup153 mRNA levels by qPCR in WT and mdx mice (n ¼ 4) and in control or LNAME-treated HL-1 (n ¼ 4). Independent Hl-1 culture was used.

Article Snippet: Lentiviral mediated sh-RNA knockdown for Nup153 was performed using 20lL of lentivirus (1 106 IFU; Santa Cruz Biotechnology sc-41279-V) dissolved in 1 mL of complete 199 medium supplemented with polybrene 5 lg/ mL.

Techniques: Expressing, Fluorescence, Control, MANN-WHITNEY

Journal: Cardiovascular research

Article Title: The nuclear pore protein Nup153 associates with chromatin and regulates cardiac gene expression in dystrophic mdx hearts.

doi: 10.1093/cvr/cvw204

Figure Lengend Snippet: Figure 2 Nitric oxide deprivation increases KAT activity and Nup153 acetylation in HL-1 cells. (A) Confocal analysis of Nup153 in HL-1 cells untreated (C) or treated with LNAME (LN, 5 mM, 24h), H2O2 (100lM, 24h), or the PCAF-specific activator SPV106 (SPV; 25lM, 4 h). Nuclei were counterstained with DAPI (scale bar: 50lm) (n ¼ 4). (B) Western blot showing Nup153 protein levels in HL-1 cells in the above conditions and densitometry analysis [*P< 0.05 vs. con- trol, t-test (n ¼ 4)]. (C) Nup153 level in HL-1 treated with LNAME alone or in combination with the KAT pan-inhibitor AA (17 lM, 24h) (n ¼ 3). Densitometry is shown in the right panel, *P < 0.05 LN vs. con- trol, **P < 0.05 LN vs. LN/AA, ANOVA followed by SNK post hoc. (D) Analysis of Nup153 acetylation level in untreated HL-1 or under NO deprivation by LNAME or oxidative stress by IP. The graph shows ace- tylated Nup153 level relative to input (n ¼ 5) *P< 0.05 vs. control, Kruskal–Wallis on ranks followed by SNK post hoc. (E) KAT activity in LNAME-treated or control HL-1 cells (0.426 0.037 control vs. 0.836 0.027 LN-treated cells, mean6 S.E.M., n ¼ 5, *P < 0.01 vs. con- trol, ANOVA, A.U. Arbitrary Units). Independent Hl-1 culture was used.

Article Snippet: Lentiviral mediated sh-RNA knockdown for Nup153 was performed using 20lL of lentivirus (1 106 IFU; Santa Cruz Biotechnology sc-41279-V) dissolved in 1 mL of complete 199 medium supplemented with polybrene 5 lg/ mL.

Techniques: Activity Assay, Western Blot, Control

Journal: Cardiovascular research

Article Title: The nuclear pore protein Nup153 associates with chromatin and regulates cardiac gene expression in dystrophic mdx hearts.

doi: 10.1093/cvr/cvw204

Figure Lengend Snippet: Figure 3 Nup153 is acetylated and associated with KATs in the mdx heart. (A) Analysis of Nup153 acetylation level by IP in WT and mdx hearts. The graph shows acetylated Nup153 level relative to input (n¼ 5, *P < 0.01 vs. WT, Mann–Whitney Rank Sum Test). (B) Co-IP showing Nup153 association with p300, PCAF, and HDAC5 in extracts from WT and mdx hearts. Densitometric analysis is shown in the right panel (n ¼ 3). (C) Evaluation of Nup153 associ- ated KAT activity in WT and mdx hearts (14.662 WT vs. 44.46 3.3 mdx, n ¼ 5 *P < 0.01 vs. WT, t-test). (D) Western blot showing Nup153 levels in WT and mdx mice treated with AA or vehicle control. Densitometry analysis is shown in the graph (n ¼ 3, *P < 0.05 vs. mdx, ANOVA followed by SNK post hoc). (E–H) KAT activity in extracts from WT and mdx organotypic cultures transduced with GFP-control or sh-Nup153 lentiviral particles for 72h in basal conditions (25.6 6 3.75WT, 54.46 3.93 mdx/LV-GFP, 38.66 1.86 mdx/LV-shNup153, *P < 0.05 vs. WT, **P < 0.05 vs. mdx/LV-GFP) (E) and after IP for PCAF (21.2 6 2.7 WT, 786 2 mdx/LV-GFP, 28.126 4.19 mdx/LV-shNup153, *P < 0.05 vs. WT, **P < 0.05 vs. mdx/LV-GFP) (F), and p300 (10.88 6 1.1 WT, 30.66 2.1 mdx/LV-GFP, 18.66 1.6 mdx/LV-shNup153, *P< 0.05 vs. WT, **P < 0.05 vs. mdx/LV-GFP) (G) (n ¼ 5, mean6 S.E.M., ANOVA fol- lowed by SNK post hoc).

Article Snippet: Lentiviral mediated sh-RNA knockdown for Nup153 was performed using 20lL of lentivirus (1 106 IFU; Santa Cruz Biotechnology sc-41279-V) dissolved in 1 mL of complete 199 medium supplemented with polybrene 5 lg/ mL.

Techniques: MANN-WHITNEY, Co-Immunoprecipitation Assay, Activity Assay, Western Blot, Control, Transduction

Journal: Cardiovascular research

Article Title: The nuclear pore protein Nup153 associates with chromatin and regulates cardiac gene expression in dystrophic mdx hearts.

doi: 10.1093/cvr/cvw204

Figure Lengend Snippet: Figure 4 Nup153 transcriptionally regulates Nexn expression in response to nitric oxide signalling in HL-1 cells. (A) Nexn mRNA level by qPCR in HL-1 treated with LNAME alone or in combination with actinomycin D (0.5lg/mL, 24h) (1.836 0.38, 4.086 0.78, 0.746 0.23 and 1.16 0.28 untreated, LNAME, ActD and LNAME/ActD, respectively), n ¼ 5, *P < 0.05 vs. control, **P < 0.01 vs. LNAME, ANOVA followed by SNK post hoc; A.U., Arbitrary Units. (B and C) ChIPs on HL1 cells treated with or without LNAME showing Nup153 binding and acetylation on histone H3 lysine 9 (H3K9ac) on Vcan and Nexn promoters (B) or on Kat8 and CycB1 promoters (C) (n ¼ 5, *P < 0.05 vs. control, ANOVA). (D) Nexn and CycB1 mRNA levels (1.96 0.31 vs. 4.026 0.63 and 6.36 1.2 vs. 7.26 1.7, respectively) assessed by qPCR, in control and LNAME-treated HL-1 cells (n ¼ 6, *P < 0.05 vs. control, ANOVA). (E) Representative western blot showing Nup153 silencing in HL-1 treated or not with LNAME for 24h. The graph shows densitometry analysis (n ¼ 5, *P < 0.05 vs. control, **P < 0.05 vs. LNAME, ANOVA followed by SNK post hoc). (F) Representative images showing Nup153 (red) and Nexilin (green) pro- teins in HL-1 cells treated with siRNA for Nup153 or scramble oligos (72 h), alone or in combination with LNAME (24 h, n ¼ 3). Nuclei were counterstained with DAPI (scale bar 50lm). Independent Hl-1 culture was used.

Article Snippet: Lentiviral mediated sh-RNA knockdown for Nup153 was performed using 20lL of lentivirus (1 106 IFU; Santa Cruz Biotechnology sc-41279-V) dissolved in 1 mL of complete 199 medium supplemented with polybrene 5 lg/ mL.

Techniques: Expressing, Control, Binding Assay, Western Blot

Journal: Cardiovascular research

Article Title: The nuclear pore protein Nup153 associates with chromatin and regulates cardiac gene expression in dystrophic mdx hearts.

doi: 10.1093/cvr/cvw204

Figure Lengend Snippet: Figure 5 Nup153 transcriptionally regulates Nexn expression in vivo. (A) mRNA evaluation of Nexn (7.86 1 WT vs. 12.66 0.9 mdx, *P < 0.05), Vcan (86 1.1 WT vs. 11.46 1.39 mdx, *P< 0.05), Crem (566 8.4 WT vs. 1026 11.3 mdx, *P < 0.05), and Adra2 (536 11.1 WT vs. 1676 40.4 mdx, *P < 0.05) in WT and mdx hearts by qPCR (sample size and mean are indicated in the figure, ANOVA). (B) Panels show representative images of Nexilin expression and distribution in WT and mdx ventricle sections co-immunostained either with actinin (upper panels) or with MHC (lower panels). Nuclei were counter- stained with DAPI (scale bar: 10lm). (C) ChIP showing Nup153 and p300 recruitment on Nexn, Kat8, and CycB promoters in WT and mdx hearts (n ¼ 6, *P < 0.05 vs. WT, t-test); signal from No antibody is also shown (NoAb). (D) Nup153 and Nexilin protein levels (left panel) or Vcan and Adra2 mRNA expression (right panel) in organotypic culture from WT and mdx hearts transduced with GFP-control or sh-Nup153 lentiviral particles (n ¼ 3, *P < 0.05 vs. WT, §P< 0.05 vs. mdx/LV-GFP, ANOVA followed by SNK post hoc).

Article Snippet: Lentiviral mediated sh-RNA knockdown for Nup153 was performed using 20lL of lentivirus (1 106 IFU; Santa Cruz Biotechnology sc-41279-V) dissolved in 1 mL of complete 199 medium supplemented with polybrene 5 lg/ mL.

Techniques: Expressing, In Vivo, Staining, Transduction, Control

Journal: Cardiovascular research

Article Title: The nuclear pore protein Nup153 associates with chromatin and regulates cardiac gene expression in dystrophic mdx hearts.

doi: 10.1093/cvr/cvw204

Figure Lengend Snippet: Figure 6 Nup153 overexpression increases Cav channel expression and function. (A) Differentiated H9c2 cells expressing either a GFP- tagged Nup153 or an empty-GFP vector, as control, analysed by west- ern blot (anti-Nup153 antibody recognizes both the endogenous and the slower migrating overexpressed GFP-tagged Nup153 protein) or by immunofluorescence. Nuclei were counterstained with DAPI (scale bar 50lm). The graph shows the percentage of transfection in the two conditions. (B) Representative traces of total (left panel) and nifedipine- sensitive Ba2þcurrent density (right panel) in control-GFP and Nup153-overexpressing H9c2 cells; (C) Mean total and Cav1 current density in GFP or Nup153 overexpressing cells (n ¼ 20, *P < 0.05 **P < 0.01, t-test). (D) Western blot showing Cav1.2 a1C subunit pro- tein levels in H9c2 cells overexpressing either GFP-tagged Nup153 or empty-GFP vectors. The graph shows the band densities (n ¼ 3, *P < 0.05 vs. empty-GFP, t-test).

Article Snippet: Lentiviral mediated sh-RNA knockdown for Nup153 was performed using 20lL of lentivirus (1 106 IFU; Santa Cruz Biotechnology sc-41279-V) dissolved in 1 mL of complete 199 medium supplemented with polybrene 5 lg/ mL.

Techniques: Over Expression, Expressing, Plasmid Preparation, Control, Immunofluorescence, Transfection, Western Blot

Journal: Cardiovascular research

Article Title: The nuclear pore protein Nup153 associates with chromatin and regulates cardiac gene expression in dystrophic mdx hearts.

doi: 10.1093/cvr/cvw204

Figure Lengend Snippet: Figure 7 Nup153 expression is altered in CMs-d-iPSC and in heart biopsies from dystrophic patients. (A) Representative images of control and BMD CMs-d-iPSC expressing cardiac Troponin T (cTnT, scale bar: 10lm). (B) Western blot showing Nup153 expression in controls (two clones), DMD (one clone), and BMD (three clones) and densitometry (mean6 S.E.M. from controls and dystrophic patients normalized vs. GAPDH, 0.446 0.06 vs. 0.896 0.03, respectively, *P < 0.01, t-test). (C) Representative images of control and BMD CMs-d-iPSC expressing Nup153 (scale bar: 5 lm) and (D) the rel- ative mean fluorescence intensity (8.226 1.38 control vs. 17.296 2.10 BMD, *P < 0.05, t-test). (E) Representative images showing Nup153 (red) and nexilin (green) expression in heart tissue samples from control (n ¼ 5) and DMD patients (n ¼ 3). Nuclei were counterstained with DAPI. Scale bar: 20lm. (F) WB showing Nup153 and nexilin levels in control and DMD heart tissue lysates (n¼ 2). (G) Representative IP showing Nup153 acetylation level in control and DMD patients (n ¼ 2).

Article Snippet: Lentiviral mediated sh-RNA knockdown for Nup153 was performed using 20lL of lentivirus (1 106 IFU; Santa Cruz Biotechnology sc-41279-V) dissolved in 1 mL of complete 199 medium supplemented with polybrene 5 lg/ mL.

Techniques: Expressing, Control, Western Blot, Clone Assay, Fluorescence

Journal: Cardiovascular research

Article Title: The nuclear pore protein Nup153 associates with chromatin and regulates cardiac gene expression in dystrophic mdx hearts.

doi: 10.1093/cvr/cvw204

Figure Lengend Snippet: Figure 8 Cartoon illustrating the proposed role of Nup153 in dys- trophic heart. In the healthy heart, NO signalling maintains low level of acetylated Nup153 favouring Nup153–HDAC5 interaction and reduc- ing the downstream target gene activation. In the dystrophic heart, NO-altered signalling affects Nup153 function inducing its association with the lysine acetylases PCAF and p300 and increasing its acetylation level. In this context, Nup153-regulated complexes may control patho- logical gene expression possibly through the binding on target pro- moters (Nexn, Crem, Adra2, Vcan).

Article Snippet: Lentiviral mediated sh-RNA knockdown for Nup153 was performed using 20lL of lentivirus (1 106 IFU; Santa Cruz Biotechnology sc-41279-V) dissolved in 1 mL of complete 199 medium supplemented with polybrene 5 lg/ mL.

Techniques: Activation Assay, Control, Gene Expression, Binding Assay

Journal: Cellular microbiology

Article Title: Incoming human papillomavirus 16 genome is lost in PML protein-deficient HaCaT keratinocytes

doi: 10.1111/cmi.12708

Figure Lengend Snippet: (A) Cells were grown on glass coverslips and infected with EdU-labeled HPV16 PsV. At 24 hpi cells were fixed and processed for the detection of EdU-labeled DNA (red), PML (green), NUP153 (blue) and DAPI (gray). (B) PML NBs and (C) EdU-labeled viral pseudogenome number was counted manually in z-stacks spanning the whole nucleus for each cell. 15 AGS cells and 13 EBV-harboring AGS cells were included in the count. P value was determined using Student's t-test. (D) RT-qPCR for viral transcription at 48 hpi with HPV16 quasivirions. cDNA samples were analyzed for HPV16E7 and the data shown are fold changes relative to AGS cells. Error bars represent SEM of five independent experiments.

Article Snippet: Antibodies and reagents Antibodies used for the study were as follows: PML (BETHYL; catalog number (#) A301-167A), lamin A/C mouse (Sigma; #SAB4200263), Nup153 (Covance; #MMS-102P), pStat1 and Stat1 (Cell Signaling; #9167S and 9176S, respectively), and β-actin (Santa Cruz; #sc-47778).

Techniques: Infection, Labeling, Quantitative RT-PCR

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: ( A ) Cartoon depicting the experimental plan for the in vitro experiments. ( B ) Schematic illustrating the main biological processes from Gene ontology analysis of Nup153-protein network in WT-NSCs

Article Snippet: Nup153 DNA from MR211997L4 plasmid was cloned by the Precision Shuttle System (Origene) in the pLenti-EF1a-C-mGFP plasmid and lentiviral particles containing GFP and Nup153 were produced by standard procedures in HEK293T cells.

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: Nup153 overexpression in AD-NSCs counteracts oxidative stress and recovers proteasomal activity. ( A ) Immunofluorescence showing GFP expression 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: Nup153 DNA from MR211997L4 plasmid was cloned by the Precision Shuttle System (Origene) in the pLenti-EF1a-C-mGFP plasmid and lentiviral particles containing GFP and Nup153 were produced by standard procedures in HEK293T cells.

Techniques: Over Expression, Activity Assay, Immunofluorescence, Expressing, 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: Nup153 overexpression in the hippocampus of AD mice stimulates proliferation and early differentiation. ( A ) Experimental plan for the in vivo experiments. ( B ) Representative confocal images showing GFP positive cells in the hippocampal niche of WT and AD mice injected with a lentivirus coding for GFP (WT and AD) or GFP-Nup153 at 10 days (T0). ( C ) Quantification of the GFP + cells in WT-GFP, AD-GFP and AD-GFP-Nup153 injected mice. Nuclei were counterstained with DAPI ( n = 3, scale bar 20 μm). ( D ) Representative confocal images showing hippocampal cells positive for BrdU (red) and DCX (green) from WT-GFP, AD-GFP and AD-GFP-Nup153 injected mice at T1. Arrows indicate BrdU/DCX double positive cells. ( E ) The graph shows the number of BrdU + , DCX + and BrdU/DCX double positive cells in WT-GFP, AD-GFP and AD-GFP-Nup153 injected mice ( n = 4, scale bar 50 μm). GCL = granule cell layer, *** P < 0.001, ** P < 0.01

Article Snippet: Nup153 DNA from MR211997L4 plasmid was cloned by the Precision Shuttle System (Origene) in the pLenti-EF1a-C-mGFP plasmid and lentiviral particles containing GFP and Nup153 were produced by standard procedures in HEK293T cells.

Techniques: Over Expression, In Vivo, Injection

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: Nup153 overexpression in the hippocampus of AD mice promotes neurogenesis and cognitive performance. ( A ) Representative confocal images showing hippocampal cells positive for BrdU (red) and P-NCAM (green) from WT-GFP, AD-GFP and AD-GFP-Nup153 injected mice at T1. Arrows indicate BrdU/P-NCAM positive cells. The graph shows the number of BrdU + , P-NCAM + and BrdU/P-NCAM double positive cells in the above conditions, n = 5 scale bar 20 μm. ( B ) Representative confocal images showing BrdU + cells (red) in the granule layer of the hippocampus identified by NeuN + cells in WT-GFP, AD-GFP and AD-GFP-Nup153 injected mice at T2 (scale bar 10 μm). The graph shows the number of BrdU/NeuN double positive cells in the hippocampus in the above conditions ( n = 4–7). ( C ) Assessment of memory performance by the Morris Water Maze test (MWM). The graph shows the time spent in the four quadrants during the probe test performed on day 5 of MWM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Nup153 DNA from MR211997L4 plasmid was cloned by the Precision Shuttle System (Origene) in the pLenti-EF1a-C-mGFP plasmid and lentiviral particles containing GFP and Nup153 were produced by standard procedures in HEK293T cells.

Techniques: Over Expression, Injection

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: Nup153 transduction in AD-iPSCs improves the maturation and organization of brain cortical organoids. ( A ) Phase contrast representative images of control, AD and AD-Nup153 organoids in the expansion phase. a1) Enlargement showing a detail of the circular neuroepithelium-like structure surrounded by the apical and basal membranes indicated by the dotted lines. ( B ) Aβ levels by dot blot analysis ( n = 3–4). Each lysate was obtained from the pool of 3–4 individual organoids. Hippocampal lysate from 9-month-old 3×Tg mice was used as positive control. Red ponceau (RP) staining was used as loading index and used to normalize samples. C-F) Confocal analysis of MAP2/Sox2 ( C and D at different magnification) and N-Cadherin ( E ) and ZO-1 ( F ) in control, AD and AD-Nup153 brain organoids. Nuclei were counterstained with DAPI, scale bar 50 μm. The inner dotted white circles indicate the ring-like N-cadherin and ZO-1 distribution at the apical membrane. The double arrow indicates the pseudostratified neuroepithelium starting from the cavity to the basal membrane. ( G ) Scheme representing the organization of the pseudostratified epithelium between the inner apical membrane and the outer basal membrane in the organoid. * P < 0.05, ** P < 0.01

Article Snippet: Nup153 DNA from MR211997L4 plasmid was cloned by the Precision Shuttle System (Origene) in the pLenti-EF1a-C-mGFP plasmid and lentiviral particles containing GFP and Nup153 were produced by standard procedures in HEK293T cells.

Techniques: Transduction, Control, Dot Blot, Positive Control, Staining, Membrane

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: 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: Nup153 DNA from MR211997L4 plasmid was cloned by the Precision Shuttle System (Origene) in the pLenti-EF1a-C-mGFP plasmid and lentiviral particles containing GFP and Nup153 were produced by standard procedures in HEK293T cells.

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

Journal: World Journal of Gastroenterology

Article Title: Quantitative proteomics analysis reveals the pathogenesis of obstructed defecation syndrome caused by abnormal expression of dystrophin

doi: 10.3748/wjg.v30.i45.4817

Figure Lengend Snippet: Primers used for polymerase chain reaction

Article Snippet: An appropriate amount of primary antibody working solution was added, including those against DMD (68120-1-Ig, Proteintech Group), NUP153 (CSB-RA986790A0HU, Cusabio), and (21774-1-AP, Proteintech Group), and incubated overnight at 4 °C.

Techniques:

Journal: World Journal of Gastroenterology

Article Title: Quantitative proteomics analysis reveals the pathogenesis of obstructed defecation syndrome caused by abnormal expression of dystrophin

doi: 10.3748/wjg.v30.i45.4817

Figure Lengend Snippet: Overexpression of nucleoporin protein 153 and L-type voltage-gated calcium channel is associated with downregulation of dystrophin expression in human intestinal smooth muscle cells. A: Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of dystrophin ( DMD ) in human intestinal smooth muscle cells transfected with negative control small interfering RNA (siR-NC) or siR-dystrophin (siR-DMD); B: RT-qPCR was used to detect the expression of nucleoporin protein 153 ( NUP153 ) in human intestinal smooth muscle cells transfected with siR-NC or siR-DMD; C: RT-qPCR was used to detect the expression of L-type voltage-gated calcium channel ( Ca v 1.2 ) in human intestinal smooth muscle cells transfected with siR-NC or siR-DMD; D: Western blot analysis of the levels of DMD, NUP153, and Ca v 1.2 in human intestinal smooth muscle cells transfected with siR-NC or siR-DMD; E: Quantitative analysis of Western blot greyscale value; F: Representative immunohistochemistry staining for NUP153 and Ca v 1.2 in rectal specimens from two groups of 50 cases. Scale bar = 50 μm; G: Quantitative analysis of the immunohistochemistry expression of NUP153 and Ca v 1.2 (integrated optical density of NUP153 and Ca v 1.2) in rectum samples of the obstructed defecation syndrome (ODS) group compared with the control group; H: Representative immunofluorescence staining for NUP153 in rectal specimens from two groups of 50 cases. Scale bar = 20 μm. Nuclei were counterstained with DAPI; I: Quantitative analysis of mean fluorescence intensity of NUP153 relative to the ODS and control groups; J: Representative immunofluorescence staining for Ca v 1.2 in rectal specimens from two groups of 50 cases. Scale bar = 20 μm. Nuclei were counterstained with DAPI; K: Quantitative analysis of mean fluorescence intensity of Ca v 1.2 relative to the ODS and control groups. a P < 0.05, c P < 0.001, d P < 0.0001, n = 3. siR-NC: Negative control small interfering RNA; siR-DMD: Dystrophin small interfering RNA; DMD: Dystrophin; ODS: Obstructed defecation syndrome; NUP153: Nucleoporin protein 153; Ca v 1.2: L-type voltage-gated calcium channel.

Article Snippet: An appropriate amount of primary antibody working solution was added, including those against DMD (68120-1-Ig, Proteintech Group), NUP153 (CSB-RA986790A0HU, Cusabio), and (21774-1-AP, Proteintech Group), and incubated overnight at 4 °C.

Techniques: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Negative Control, Small Interfering RNA, Western Blot, Immunohistochemistry, Staining, Control, Immunofluorescence, Fluorescence

Journal: World Journal of Gastroenterology

Article Title: Quantitative proteomics analysis reveals the pathogenesis of obstructed defecation syndrome caused by abnormal expression of dystrophin

doi: 10.3748/wjg.v30.i45.4817

Figure Lengend Snippet: Diagram illustrating role of dystrophin in obstructed defecation syndrome (created with Figdraw 2.0). In intestinal smooth muscle cells, altered expression of dystrophin affects the expression of nucleoporin protein 153, leading to its L-type voltage-gated calcium channel being overactivated and increasing calcium influx. Under these circumstances, the contractile function of intestinal smooth muscle cells is impaired, resulting in insufficient rectal propulsion force, ultimately leading to obstructed defecation syndrome. ODS: Obstructed defecation syndrome; NUP153: Nucleoporin protein 153; Ca v 1.2: L-type voltage-gated calcium channel.

Article Snippet: An appropriate amount of primary antibody working solution was added, including those against DMD (68120-1-Ig, Proteintech Group), NUP153 (CSB-RA986790A0HU, Cusabio), and (21774-1-AP, Proteintech Group), and incubated overnight at 4 °C.

Techniques: Expressing