Journal: Epigenetics & Chromatin

Article Title: HOXA repression is mediated by nucleoporin Nup93 assisted by its interactors Nup188 and Nup205

doi: 10.1186/s13072-016-0106-0

Figure Lengend Snippet: Nup93 interacts with Nup188 and Nup205 and associates with the HOXA1 promoter. a , b Immunoprecipitation was performed using antibodies specific for ( a ) Nup93; ( b ) Nup188 and IgG followed by Western blotting for Nup93, Nup188, Nup205 and negative control—Nup98 (representative data from three independent biological replicates, N = 3, single experiment for Nup98). c ( i ) Nup188 and ( ii ) Nup205 were knocked down in DLD1 cells using siRNA. A representative Western blot showing the extent of knockdown (representative Western blot from three independent biological replicates, N = 3). d ChIP experiment was performed using an anti-Nup93 antibody in untreated, non-targeting siRNA control (siNeg), Nup188 Kd (Knockdown) and Nup205 Kd cells. ChIP-qPCR analysis was used to determine the extent of Nup93 association with the HOXA1 promoter in Nup188 and Nup205 knockdown cells (Input and PanH3 in Fig. 2d are from Nup205 Kd sample) Y -axis: immunoprecipitated DNA relative to 1% input, corrected for ChIP using non-specific IgG ( N = 2, data from two independent biological replicates that include a total of six technical replicates), error bar : standard error of mean (SEM). e A representative Western blot showing the effect of Nup93, Nup188 and Nup205 depletion on one another (three independent biological replicates, N = 3), f , g a representative Western blot showing overexpression of Nup93 upon Nup188 ( f ) and Nup205 knockdown ( g ). GAPDH was used as a loading control. h , i ChIP-qPCR was performed upon overexpression of ( f ) Nup93 in Nup188- and ( g ) Nup205-depleted cells. Y -axis: immunoprecipitated DNA relative to 1% input, corrected for ChIP using non-specific IgG ( N = 2, data from two independent biological replicates that include a total of six technical replicates), error bar : standard error of mean (SEM)

Article Snippet: Rabbit anti-Nup93 (1:500, sc-292099, Lot-E0211, Santa Cruz Biotechnology, CA), rabbit anti-Nup188 (1:1000, Abcam, ab86601, Lot-GR43443-4), mouse anti-Nup98 (1:500, sc-74553, Lot-H0108, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Nup205 antibody (1:500, HPA024574, Lot-R11937, Atlas Antibodies), rabbit anti-EED (1:500, ab4469, Lot-GR51357-1), rabbit anti-EZH2 (1:500, ab3748, Lot-GR252135-1), rabbit anti-Suz12 (1:500, ab12073, Lot-GR79631-1) and rabbit anti-CTCF antibody (1:500, 07-729, Lot-2375606, Millipore).

Techniques: Immunoprecipitation, Western Blot, Negative Control, Knockdown, Control, ChIP-qPCR, Over Expression

Journal: Epigenetics & Chromatin

Article Title: HOXA repression is mediated by nucleoporin Nup93 assisted by its interactors Nup188 and Nup205

doi: 10.1186/s13072-016-0106-0

Figure Lengend Snippet: Depletion of Nup93, Nup188 and Nup205 derepresses HOXA gene cluster. a – c qRT-PCR analyses was used to determine mRNA levels of all HOXA genes (HOXA1 to HOXA13) upon ( a ) Nup93, ( b ) Nup188 and ( c ) Nup205 knockdowns in DLD1 cells. Graph represents fold change ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2^{{ - {\Delta \Delta }C_{\text{t}} }}$$\end{document} 2 - Δ Δ C t ) in levels of mRNA normalized to untreated cells. Error bars: SEM, data from three independent biological replicates that include total of nine technical replicates, * p < 0.05; ** p < 0.01; *** p < 0.001 (Students t test between siNeg and knockdown). GLCCI, served as a negative control. d , e qRT-PCR analyses was used to determine mRNA levels of all HOXA genes (HOXA1 to HOXA13) upon Nup93 overexpression in ( d ) Nup188- and ( e ) Nup205-depleted cells. Graph represents fold change ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2^{{ - {\Delta \Delta }C_{\text{t}} }}$$\end{document} 2 - Δ Δ C t ) in levels of mRNA normalized to untreated cells. Error bars : SEM, data from two independent biological replicates that include total of six technical replicates. GLCCI, served as a negative control

Article Snippet: Rabbit anti-Nup93 (1:500, sc-292099, Lot-E0211, Santa Cruz Biotechnology, CA), rabbit anti-Nup188 (1:1000, Abcam, ab86601, Lot-GR43443-4), mouse anti-Nup98 (1:500, sc-74553, Lot-H0108, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Nup205 antibody (1:500, HPA024574, Lot-R11937, Atlas Antibodies), rabbit anti-EED (1:500, ab4469, Lot-GR51357-1), rabbit anti-EZH2 (1:500, ab3748, Lot-GR252135-1), rabbit anti-Suz12 (1:500, ab12073, Lot-GR79631-1) and rabbit anti-CTCF antibody (1:500, 07-729, Lot-2375606, Millipore).

Techniques: Quantitative RT-PCR, Knockdown, Negative Control, Over Expression

Journal: Epigenetics & Chromatin

Article Title: HOXA repression is mediated by nucleoporin Nup93 assisted by its interactors Nup188 and Nup205

doi: 10.1186/s13072-016-0106-0

Figure Lengend Snippet: HOXA gene loci is untethered from the nuclear periphery upon Nup93, Nup188 and Nup205 depletion. a Representative images (maximum intensity projection of a confocal image stack) of 3D-FISH for HOXA ( red ), CT7 ( green ) and DAPI ( blue ) performed on siLacZ-, Nup93-, Nup188-, Nup205- and Nup98-depleted DLD1 cells. Scale bar ~10 μm, white dotted line indicates nuclear boundary. b Dot scatter plot showing shortest distance of HOXA gene locus from nuclear periphery demarcated by DAPI in siLacZ ( n = 164 loci signals)-, Nup93 ( n = 154)-, Nup188 ( n = 178)-, Nup205 ( n = 178)- and Nup98 ( n = 124)-depleted DLD1 cells, horizontal bar represents median with interquartile range. Data from two independent biological replicates, ** p < 0.01; **** p < 0.001 (Kolmogorov–Smirnov test). c % Frequency distribution profile of HOXA gene locus from nuclear periphery plotted as bins of ~0.2 µm each from the nuclear periphery. Y -axis represents % frequency of HOXA locus pooled from two independent biological replicates

Article Snippet: Rabbit anti-Nup93 (1:500, sc-292099, Lot-E0211, Santa Cruz Biotechnology, CA), rabbit anti-Nup188 (1:1000, Abcam, ab86601, Lot-GR43443-4), mouse anti-Nup98 (1:500, sc-74553, Lot-H0108, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Nup205 antibody (1:500, HPA024574, Lot-R11937, Atlas Antibodies), rabbit anti-EED (1:500, ab4469, Lot-GR51357-1), rabbit anti-EZH2 (1:500, ab3748, Lot-GR252135-1), rabbit anti-Suz12 (1:500, ab12073, Lot-GR79631-1) and rabbit anti-CTCF antibody (1:500, 07-729, Lot-2375606, Millipore).

Techniques:

Journal: Epigenetics & Chromatin

Article Title: HOXA repression is mediated by nucleoporin Nup93 assisted by its interactors Nup188 and Nup205

doi: 10.1186/s13072-016-0106-0

Figure Lengend Snippet: Nup93 depletion reduces nuclear import but does not affect nuclear export. a A representative image of nuclear import assay performed using GR2-GFP2-M9 construct transfected in cells treated independently with LacZ and siNup93. To induce nuclear import of GR2-GFP2-M9 fusion protein, cells were treated with dexamethasone (Dex) (5 µM) for 30 min, white arrowhead indicates absence of cytoplasmic GFP in LacZ + Dex and residual cytoplasmic GFP in Nup93 Kd + Dex. Scale bar ~10 µm. b A representative image of Poly(A) RNA FISH performed using FAM-labeled oligo(dT) probe ( green ) in siNeg, Nup93 Kd, Nup188 Kd, Nup205 Kd and Nup98 Kd, scale bar ~10 µm, white arrowhead indicates Poly(A) RNA foci in the nucleus. Nuclear boundary is marked by dotted line in enlarged panel. Nup98 enlarged panel shows both nuclear and cell boundary with white dotted line. c Nuclear/cytoplasmic (N/C) ratio of GR2-GFP2-M9 was determined by quantifying its relative fluorescence intensity in the nucleus and cytoplasm. Scatter plot of GFP signals expressed as nuclear-to-cytoplasmic ratios from LacZ ( n = 60 cells), Nup93 Kd ( n = 57), Nup188 Kd ( n = 60), Nup205 Kd ( n = 59) and Nup98 Kd ( n = 60), data from 2 independent biological replicates (**** p < 0.0001). d Poly(A) RNA distribution was determined by quantifying its fluorescence intensity in the nucleus and cytoplasm. Scatter plot of Poly(A) signals expressed as nuclear (N)-to-cytoplasmic (C) ratios from siNeg ( n = 127 cells), Nup93 Kd ( n = 158), Nup188 Kd ( n = 288), Nup205 Kd ( n = 288); N/C ratio was not significant (ns) when compared to siNeg ( p > 0.05), while Nup98 Kd ( n = 97) shows a relatively higher nuclear-to-cytoplasmic ratio (N/C ratio) of Poly(A) signals (*** p = 0.0017). Two independent biological replicates for siNeg, Nup93 Kd and Nup98 Kd. Data from a single experiment for Nup188 Kd and Nup205 Kd. Horizontal line represents median, p values obtained from Mann–Whitney U test. Nuclear transport of Poly(A) RNA was unaffected in Nup93-, Nup188- or Nup205- depleted cells

Article Snippet: Rabbit anti-Nup93 (1:500, sc-292099, Lot-E0211, Santa Cruz Biotechnology, CA), rabbit anti-Nup188 (1:1000, Abcam, ab86601, Lot-GR43443-4), mouse anti-Nup98 (1:500, sc-74553, Lot-H0108, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Nup205 antibody (1:500, HPA024574, Lot-R11937, Atlas Antibodies), rabbit anti-EED (1:500, ab4469, Lot-GR51357-1), rabbit anti-EZH2 (1:500, ab3748, Lot-GR252135-1), rabbit anti-Suz12 (1:500, ab12073, Lot-GR79631-1) and rabbit anti-CTCF antibody (1:500, 07-729, Lot-2375606, Millipore).

Techniques: Construct, Transfection, Labeling, Fluorescence, MANN-WHITNEY

Journal: Epigenetics & Chromatin

Article Title: HOXA repression is mediated by nucleoporin Nup93 assisted by its interactors Nup188 and Nup205

doi: 10.1186/s13072-016-0106-0

Figure Lengend Snippet: HOXA is upregulated upon Nup93 depletion independent of CTCF. a Co-immunoprecipitation was performed using anti-Nup93 antibody and negative control IgG followed by Western blotting for CTCF, Nup205, Nup188 and Nup93 (data from two independent biological replicates, N = 2), b Co-IP for Nup93 and Western blot for Nup93, Nup188, Nup205 (data from three independent biological replicates, N = 3), PRC2 complex proteins EED and Suz12 (data from a single experiment). c Representative Western blot showing the levels of Nup93, Nup188, Nup205, Nup98, EZH2, Suz12, CTCF, EED upon Nup93, Nup188 and Nup205 Kd (data from a single experiment). d Representative Western blot showing siRNA-mediated knockdown of CTCF in DLD1 cells. e Epigenome Browser view of CTCF (GSM749729) ( arrow indicates potential binding sites of Nup93) on HOXA gene cluster. f qRT-PCR analysis was used to determine mRNA levels of all HOXA genes (HOXA1 to HOXA13) upon CTCF and combined Nup93 + CTCF knockdowns in DLD1 cells. Graph represents fold change ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2^{{ - {\Delta \Delta }C_{\text{t}} }}$$\end{document} 2 - Δ Δ C t ) in levels of mRNA normalized to untreated cells. Error bars SEM, data from two independent biological replicates ( N = 2) that includes total of 6 technical replicates, GLCCI, served as control. Nup93 Kd data ( green bars ) is from Fig. a, plotted here for comparison between Nup93 Kd with Nup93 + CTCF Kd ( orange bars ). Nup93 does not interact with CTCF or PRC2 complex proteins. Nup93 Kd upregulates HOXA gene expression independent of CTCF. CTCF depletion alone upregulates GLCCI1, which is unaffected upon Nup93 knockdown

Article Snippet: Rabbit anti-Nup93 (1:500, sc-292099, Lot-E0211, Santa Cruz Biotechnology, CA), rabbit anti-Nup188 (1:1000, Abcam, ab86601, Lot-GR43443-4), mouse anti-Nup98 (1:500, sc-74553, Lot-H0108, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Nup205 antibody (1:500, HPA024574, Lot-R11937, Atlas Antibodies), rabbit anti-EED (1:500, ab4469, Lot-GR51357-1), rabbit anti-EZH2 (1:500, ab3748, Lot-GR252135-1), rabbit anti-Suz12 (1:500, ab12073, Lot-GR79631-1) and rabbit anti-CTCF antibody (1:500, 07-729, Lot-2375606, Millipore).

Techniques: Immunoprecipitation, Negative Control, Western Blot, Co-Immunoprecipitation Assay, Knockdown, Binding Assay, Quantitative RT-PCR, Control, Comparison, Gene Expression

Journal: Cell reports

Article Title: Acute depletion of human core nucleoporin reveals direct roles in transcription control but dispensability for 3D genome organization

doi: 10.1016/j.celrep.2022.111576

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal NUP205 Antibody , Bethyl Laboratories , Cat#A303-935, RRID: AB_2620284.

Techniques: Recombinant, SYBR Green Assay, Protease Inhibitor, Magnetic Beads, Lysis, Extraction, cDNA Synthesis, Bicinchoninic Acid Protein Assay, HiChIP, Gene Expression, Generated, Expressing, Software, CRISPR