Journal: Journal of proteome research

Article Title: Proteomic Profile Identifies Dysregulated Pathways in Cornelia de Lange Syndrome Cells With Distinct Mutations in SMC1A and SMC3 Genes

doi: 10.1021/pr300760p

Figure Lengend Snippet: Analysis of cohesin binding at the human c-MYC locus. (A) Schematic of human c-MYC gene. Black solid boxes indicate translated regions. Cohesin binding to MINE and first exon is denoted by an asterisk. Arrows indicate transcriptional start sites of the P1 and P2 promoter. Location of primer pairs A-C are indicated, ChIP assays with antibody against RAD21 were analysed by real-time PCR with primers pairs B and C specific for the promoter region of the c-MYC locus. Binding at each site was determined relative to primer A, where no RAD21 binding was predicted. (B) Results showed that RAD21 co-localized to the promoter region of c-MYC in all SMC1A- and SMC3-mutated CdLS cell lines, with the exception of CdL057, whereas bound cohesin was dramatically reduced in exon 1 in CdLVH, CdLSS, CdL057, CdL060, CdL107 CdLS cell lines. CdL203, which shares the same mutation with CdL060, showed a decrease close to significant (p = 0.07). Since no difference was found in control cell lines, data was pooled. Results shown are the averages of three independent experiments. The graphs show the average and the standard error of the normalized values and *p < 0.05.

Article Snippet: Antibody against RAD21 (Bethyl Laboratories) was used.

Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Mutagenesis, Control

Journal: Journal of proteome research

Article Title: Proteomic Profile Identifies Dysregulated Pathways in Cornelia de Lange Syndrome Cells With Distinct Mutations in SMC1A and SMC3 Genes

doi: 10.1021/pr300760p

Figure Lengend Snippet: Mutated SMC1A and SMC3 co-immunoprecipitate with RAD21 in the CdLS cell lines. (A) SMC1A (and SMC3) was found to be co-precipitated with RAD21, (B) whereas no RAD21 signal was detected in the IPs using IgG-coated beads. (C) In addition, RAD21co-precipitated with SMC1A (and SMC3) and (D) no SMC specific signal was detectable in the IPs using IgG-coated beads.

Article Snippet: Antibody against RAD21 (Bethyl Laboratories) was used.

Techniques:

Journal: Frontiers in Oncology

Article Title: RAD21 Confers Poor Prognosis and Affects Ovarian Cancer Sensitivity to Poly(ADP-Ribose)Polymerase Inhibitors Through DNA Damage Repair

doi: 10.3389/fonc.2022.936550

Figure Lengend Snippet: RAD21 expression level in different groups of ovarian tissue and its prognostic value in epithelial ovarian cancer. (A) Representative images of immunohistochemical staining for RAD21 in ovarian tissue samples. Magnification: upper, ×200; lower, ×400. (B) Immunostaining scores of RAD21 in ovarian tissue samples. (C) Statistical analyses of RAD21-positive and high-positive rates in ovarian tissue samples. (D) The relationship between RAD21 expression, International Federation of Gynecology and Obstetrics (FIGO) stage, lymph node metastasis, and overall survival in patients with epithelial ovarian cancer. (E) Forest plot based on Cox regression model analysis of patients with epithelial ovarian cancer. * p < 0.05; ** p < 0.01;*** p < 0.001; ns, not significant.

Article Snippet: The concentration of the polyclonal antibody used against RAD21 was 1:250 (27071-1-AP; ProteinTech, Wuhan, China).

Techniques: Expressing, Immunohistochemical staining, Staining, Immunostaining

Journal: Frontiers in Oncology

Article Title: RAD21 Confers Poor Prognosis and Affects Ovarian Cancer Sensitivity to Poly(ADP-Ribose)Polymerase Inhibitors Through DNA Damage Repair

doi: 10.3389/fonc.2022.936550

Figure Lengend Snippet: Relationship between RAD21 expression level in epithelial ovarian cancer and clinicopathological parameters.

Article Snippet: The concentration of the polyclonal antibody used against RAD21 was 1:250 (27071-1-AP; ProteinTech, Wuhan, China).

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: RAD21 Confers Poor Prognosis and Affects Ovarian Cancer Sensitivity to Poly(ADP-Ribose)Polymerase Inhibitors Through DNA Damage Repair

doi: 10.3389/fonc.2022.936550

Figure Lengend Snippet: RAD21 overexpression promoted proliferation, migration, and invasion of ovarian cancer cells. (A) RAD21 expression after transfection with siRNA or lentivirus. (B) Ovarian cancer cell post-transfection proliferation ability evaluated using Cell Counting Kit-8 (CCK8) assays. (C) Ovarian cancer cell post-transfection migration ability evaluated using wound-healing assays. (D) Ovarian cancer cell post-transfection invasion ability evaluated using invasion assays. (E) The expression of matrix metalloproteinase 2 (MMP2) and MMP9 after transfection measured using Western blotting. Data were represented as mean ± SD (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The concentration of the polyclonal antibody used against RAD21 was 1:250 (27071-1-AP; ProteinTech, Wuhan, China).

Techniques: Over Expression, Migration, Expressing, Transfection, Cell Counting, Western Blot

Journal: Frontiers in Oncology

Article Title: RAD21 Confers Poor Prognosis and Affects Ovarian Cancer Sensitivity to Poly(ADP-Ribose)Polymerase Inhibitors Through DNA Damage Repair

doi: 10.3389/fonc.2022.936550

Figure Lengend Snippet: RAD21 overexpression reduced poly(ADP-ribose)polymerase (PARP) inhibitor sensitivity in ovarian cancer cells. (A–C) Cell viability in cells treated with olaparib (A) , rucaparib (B) , and niraparib (C) analyzed using the Cell Counting Kit-8 (CCK8) assay. (D) IC 50 values in cells treated with PARP inhibitors. (E, F) The effects of RAD21 on olaparib treated OVCAR3 (E) and ES-2 (F) cell proliferation. (G, H) The effects of RAD21 on rucaparib-treated OVCAR3 (G) and ES-2 (H) cell proliferation. (I, J) The effects of RAD21 on niraparib-treated OVCAR3 (I) and ES-2 (J) cell proliferation. Data were represented as mean ± SD (n = 3). * p < 0.05; ** p < 0.01.

Article Snippet: The concentration of the polyclonal antibody used against RAD21 was 1:250 (27071-1-AP; ProteinTech, Wuhan, China).

Techniques: Over Expression, Cell Counting, CCK-8 Assay

Journal: Frontiers in Oncology

Article Title: RAD21 Confers Poor Prognosis and Affects Ovarian Cancer Sensitivity to Poly(ADP-Ribose)Polymerase Inhibitors Through DNA Damage Repair

doi: 10.3389/fonc.2022.936550

Figure Lengend Snippet: Effects of RAD21 on DSB repair after treatment with poly(ADP-ribose)polymerase (PARP) inhibitors. (A–C) Representative images of immunofluorescence staining for γ-H2AX and numerical quantification of γ-H2AX foci in ovarian cancer cells treated with olaparib (A) , rucaparib (B) , and niraparib (C) . Scale bar = 10 μm. Data were represented as mean ± SD (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The concentration of the polyclonal antibody used against RAD21 was 1:250 (27071-1-AP; ProteinTech, Wuhan, China).

Techniques: Immunofluorescence, Staining

Journal: Frontiers in Oncology

Article Title: RAD21 Confers Poor Prognosis and Affects Ovarian Cancer Sensitivity to Poly(ADP-Ribose)Polymerase Inhibitors Through DNA Damage Repair

doi: 10.3389/fonc.2022.936550

Figure Lengend Snippet: RAD21 regulated ovarian cancer progression by activating the Akt/mTOR signaling pathway. (A) Western blotting analysis showing the levels of Akt, p-Akt, mTOR, and p-mTOR in ovarian cancer cells with RAD21 knockdown or overexpression. (B) Wound-healing assay showing the migration ability of ovarian cancer cells treated with PP242. (C) Invasion assay showing the invasion ability of ovarian cancer cells treated with PP242. (D) Cell Counting Kit-8 (CCK8) assay showing the proliferation ability of ovarian cancer cells treated with PP242. (E) Cell viability of ovarian cancer cells treated with PP242 and poly(ADP-ribose)polymerase (PARP) inhibitors. Data were represented as mean ± SD (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The concentration of the polyclonal antibody used against RAD21 was 1:250 (27071-1-AP; ProteinTech, Wuhan, China).

Techniques: Western Blot, Knockdown, Over Expression, Wound Healing Assay, Migration, Invasion Assay, Cell Counting, CCK-8 Assay

Journal: Frontiers in Oncology

Article Title: RAD21 Confers Poor Prognosis and Affects Ovarian Cancer Sensitivity to Poly(ADP-Ribose)Polymerase Inhibitors Through DNA Damage Repair

doi: 10.3389/fonc.2022.936550

Figure Lengend Snippet: The impact of RAD21 on tumor formation in vivo . (A) Subcutaneous xenograft nude mouse model. (B) Images of tumors from nude mice in the vector and RAD21-overexpressing groups. (C) Subcutaneous tumor volume in the two groups. (D) Tumor weight in the two groups. (E) H&E and immunohistochemical staining in the two groups. * p < 0.05; ** p < 0.01.

Article Snippet: The concentration of the polyclonal antibody used against RAD21 was 1:250 (27071-1-AP; ProteinTech, Wuhan, China).

Techniques: In Vivo, Plasmid Preparation, Immunohistochemical staining, Staining

Journal: Frontiers in Oncology

Article Title: RAD21 Confers Poor Prognosis and Affects Ovarian Cancer Sensitivity to Poly(ADP-Ribose)Polymerase Inhibitors Through DNA Damage Repair

doi: 10.3389/fonc.2022.936550

Figure Lengend Snippet: A schematic diagram illustrating the role of RAD21 in ovarian cancer.

Article Snippet: The concentration of the polyclonal antibody used against RAD21 was 1:250 (27071-1-AP; ProteinTech, Wuhan, China).

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells

doi: 10.3389/fmolb.2021.757394

Figure Lengend Snippet: Gene targeting efficiencies in mESC and HAP1 clones in the example of the AtAFB2 system.

Article Snippet: After blocking in 5% milk/TBST for 2 h, the membrane was incubated with primary antibodies against RAD21 and SMC2 (#12673/#8720, Cell Signaling Technology) at 4°C overnight.

Techniques: Clone Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells

doi: 10.3389/fmolb.2021.757394

Figure Lengend Snippet: Haploid and diploid HAP1 clones could be reliably separated by cell size using FACS or visual clues. (A) FACS plots for haploid and diploid HAP1 clones. (B) Mosaic HAP1 clone consisting of two subpopulations in equal proportions. Blue/red highlight indicates haploid/diploid subpopulations, respectively. (C) Two eGFP-positive RAD21-miniIAA7-eGFP clones that show significant cell size differences, making it easy to pick the correct clone (haploid clone is on the right).

Article Snippet: After blocking in 5% milk/TBST for 2 h, the membrane was incubated with primary antibodies against RAD21 and SMC2 (#12673/#8720, Cell Signaling Technology) at 4°C overnight.

Techniques: Clone Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells

doi: 10.3389/fmolb.2021.757394

Figure Lengend Snippet: Degradation efficiency and dynamics of POI depletion in mESC and HAP1 clones with randomly integrated OsTIR1 and AtAFB2 constructs. POI degradation efficiency in mESC clones (A) and HAP1 clones (B) . Each dot represents one independent cell clone derived after AFB protein gene integration. For each clone, the measurement was carried out once. The black horizontal line shows the median of the group. (C) POI degradation dynamics for selected mESC clones (Rad21-mAID-Clover, Smc2-mAID-Clover, Rad21-miniIAA7-eGFP, Smc2-miniIAA7-eGFP) with OsTIR1 and AtAFB2. For each clone, the experiment was carried out once. (D) POI degradation dynamics for HAP1 clones (RAD21-miniIAA7-eGFP, RAD21-mAID-Clover) with AtAFB2 and OsTIR1. For each clone, the experiment was carried out once. (E) POI degradation dynamics for HAP1 clones (SMC2-miniIAA7-eGFP, SMC2-mAID-Clover) with AtAFB2 and OsTIR1. Numbers in brackets are individual clones’ designations. For each clone, the experiment was carried out once.

Article Snippet: After blocking in 5% milk/TBST for 2 h, the membrane was incubated with primary antibodies against RAD21 and SMC2 (#12673/#8720, Cell Signaling Technology) at 4°C overnight.

Techniques: Clone Assay, Construct, Derivative Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells

doi: 10.3389/fmolb.2021.757394

Figure Lengend Snippet: Comparison of degradation efficiency with AFB protein driven by the same EF1a promoter. (A) Scheme of genetic constructs used. (B) Dynamics of POI depletion in mESC clones (Rad21 colored in red; Smc2 colored in blue). Vectors expressing AFB proteins: pSH-EFIRES-P-OsTIR1-mCherry-weak NLS and pSH-EFIRES-P-AtAFB2-mCherry-weak NLS, were randomly integrated. For this analysis, all clones were grown in puromycin, and the proportion of Cherry-positive cells in each of them was more than 95%. For each clone, the measurement was carried out twice.

Article Snippet: After blocking in 5% milk/TBST for 2 h, the membrane was incubated with primary antibodies against RAD21 and SMC2 (#12673/#8720, Cell Signaling Technology) at 4°C overnight.

Techniques: Comparison, Construct, Clone Assay, Expressing

Journal: Frontiers in Molecular Biosciences

Article Title: Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells

doi: 10.3389/fmolb.2021.757394

Figure Lengend Snippet: Effects of pH on AtAFB2 and OsTIR1 AID degradation efficiency. (A) High pH levels inhibit auxin-induced eGFP degradation in HAP1 clones with RAD21 and SMC2 modifications. Experiments were performed in duplicate. (B) IAA and NAA auxin analogs show similar sensitivity to pH changes (HAP1 clones). Experiments were performed in duplicate. (C) eGFP levels in the mosaic HAP1 clone SMC2-miniIAA7-eGFP. Blue color indicates eGFP levels in Cherry pos subpopulation, and purple color indicates Cherry neg subpopulation. The shift between two peaks illustrates minor basal degradation induced by AtAFB2 in Cherry pos cells. (D) Shift of mES cell population (OsTIR1 system) toward left on the FITC-A axis in acidic media.

Article Snippet: After blocking in 5% milk/TBST for 2 h, the membrane was incubated with primary antibodies against RAD21 and SMC2 (#12673/#8720, Cell Signaling Technology) at 4°C overnight.

Techniques: Clone Assay

Journal: Science Advances

Article Title: ASXL1 interacts with the cohesin complex to maintain chromatid separation and gene expression for normal hematopoiesis

doi: 10.1126/sciadv.1601602

Figure Lengend Snippet: ( A ) Table of the most relevant proteins identified by LC-MS/MS in the affinity purification of ASXL1-associated proteins using FLAG-ASXL1 overexpressing HEK293T cells. Spectral counts (unique and total) for each interacting protein are shown. ( B to E ) Reciprocal IP and Western blotting confirmed interaction of ASXL1 with SMC1A, SMC3, and RAD21 in nuclear fraction derived from HEK293T cells transfected with pcDNA3.1 + (Vec) or FLAG-tagged ASXL1 (ASXL1). Nuclear extractions were subjected to IP using indicated antibodies against FLAG (B), SMC1A (C), SMC3 (D), or RAD21 (E). IB, immunoblot. ( F ) Western blot shows the endogenous interaction between ASXL1 and SMC1A, SMC3, and RAD21 in BM cells of WT mice. IgG, immunoglobulin G. ( G ) Gel filtration analysis of nuclear extracts from FLAG-ASXL1 overexpressing cells. ASXL1 and the cohesin complex were coeluted from a Superose 6 HR gel filtration column, as analyzed by Western blotting. The numbers over the lanes represent the eluted fraction numbers.

Article Snippet: Genomic DNA regions of interest were isolated using antibodies against ASXL1 (Santa Cruz Biotechnology, sc-85283), SMC1A (Abcam, ab133643), and RAD21 (Abcam, ab154769).

Techniques: Liquid Chromatography with Mass Spectroscopy, Affinity Purification, Western Blot, Derivative Assay, Transfection, Filtration

Journal: Science Advances

Article Title: ASXL1 interacts with the cohesin complex to maintain chromatid separation and gene expression for normal hematopoiesis

doi: 10.1126/sciadv.1601602

Figure Lengend Snippet: ( A ) Schematic diagram of the full-length (FL) ASXL1 and the truncated variants of Asxl1 [amino acids (aa) 1 to 1010, 1 to 420, 1 to 587, and 401 to 587]. Binding affinity was determined by the pull-down efficiency of IP with anti-FLAG and Western blotting with cohesin antibodies. NLS, nuclear localization signal. ( B to E ) Western blotting analysis of nuclear fractions and anti-FLAG immunoprecipitates from pcDNA3.1 + , or each truncated ASXL1 transfected HEK293T cells using antibodies against FLAG, SMC1A, SMC3, or RAD21.

Article Snippet: Genomic DNA regions of interest were isolated using antibodies against ASXL1 (Santa Cruz Biotechnology, sc-85283), SMC1A (Abcam, ab133643), and RAD21 (Abcam, ab154769).

Techniques: Binding Assay, Western Blot, Transfection

Journal: Science Advances

Article Title: ASXL1 interacts with the cohesin complex to maintain chromatid separation and gene expression for normal hematopoiesis

doi: 10.1126/sciadv.1601602

Figure Lengend Snippet: ( A and B ) The myeloid cells with premature sister chromatid separation are frequently seen in PB smears (A) and BM (B) of Asxl1 +/− and Asxl1 −/− mice with MDS. Red arrows indicate the abnormal nuclear bridging. Scale bars, 5 μm (A) and 10 μm (B). ( C and D ) Representative cells with premature sister chromatid separation in cultured WT, Asxl1 +/− , and Asxl1 −/− LK cells. Red arrows indicate the premature sister chromatid separation. The frequency of cells with premature sister chromatid separation is shown in (C). Y axis shows the percentage of cells with premature sister chromatid separation within all binucleated cells. Data are represented as means ± SEM from three independent experiments. *** P < 0.001 and ** P < 0.01. Scale bars, 5 μm. ( E ) The frequency of cells with premature sister chromatid separation in the HeLa GFP-H2B cells with hASXL1 KD and hASXL1 KD plus mAsxl1 rescues. KD of ASXL1 leads to increased frequency of cells with premature sister chromatid separation in HeLa GFP-H2B cells. Reintroducing full-length mAsxl1 rescued the premature sister chromatid separation in HeLa cells with ASXL1 KD. Data are represented as means ± SEM from three independent experiments. *** P < 0.001 and ** P < 0.01. ( F and G ) SMC1A or RAD21 KD leads to premature sister chromatid separation in HeLa GFP-H2B cells. Representative photomicrographs show the cells with premature sister chromatid separation, as indicated by red arrowheads (G). The frequency of cells with premature sister chromatid separation is shown in (F). Y axis shows the percentage of cells with premature sister chromatid separation within all binucleated cells. Data are represented as means ± SEM from three independent experiments. *** P < 0.001 and ** P < 0.01. Scale bars, 5 μm. ( H ) Western blotting shows the expression of full-length ASXL1 and ASXL1 amino acids 401 to 587 in HeLa GFP-H2B cells transfected with vector only, full-length ASXL1, or ASXL1 amino acids 401 to 587. β-Actin serves as loading control. ( I and J ) ASXL1 amino acids 401 to 587 induce chromatin bridging in HeLa GFP-H2B cells. Quantification of the frequency of cells with premature sister chromatid separation in HeLa GFP-H2B cells transfected with pcDNA3.1 + , full-length ASXL1, or ASXL1 amino acids 401 to 587 (J). Data are represented as means ± SEM from three independent experiments. ** P < 0.01 for ASXL1 amino acids 401 to 587 fragment versus pcDNA3.1 + or full-length ASXL1. Red arrows indicate the premature sister chromatid separation. Scale bars, 5 μm.

Article Snippet: Genomic DNA regions of interest were isolated using antibodies against ASXL1 (Santa Cruz Biotechnology, sc-85283), SMC1A (Abcam, ab133643), and RAD21 (Abcam, ab154769).

Techniques: Cell Culture, Western Blot, Expressing, Transfection, Plasmid Preparation

Journal: Science Advances

Article Title: ASXL1 interacts with the cohesin complex to maintain chromatid separation and gene expression for normal hematopoiesis

doi: 10.1126/sciadv.1601602

Figure Lengend Snippet: ( A ) Venn diagram showing overlapping peaks between ASXL1, SMC1A, and RAD21 ChIP-seq in WT LK cells. ( B ) Genomic distribution of ASXL1/SMC1A/RAD21 triple overlapping ChIP peaks in WT LK cells. ( C ) The overlap analysis shows the peak reads in WT and Asxl1 −/− LK cells based on ASXL1/SMC1A/RAD21 overlapping peaks from WT LK cells. Zero base pair (bp) is defined as the peak of ASXL1 binding sites on the genome of WT LK cells. Decreased genomic cohesin complex occupancy is seen in Asxl1 −/− LK cells. The overlap peaks of SMC1A and RAD21 represent the cohesin occupancy on the genome. Comparison of the SMC1A/RAD21 overlapping peaks between WT and Asxl1 −/− LK cells identified 7833-peak loss and 1175-peak gain in the Asxl1 −/− LK cells. TSS, transcription start site. ( D ) The pie chart represents the percentage of genes with no cohesin occupancy change (remain) or cohesin loss (SMC1A and/or RAD21 peak loss) in Asxl1 −/− BM LK cells based on all ASXL1/SMC1A/RAD21 triple overlapping peaks of WT LK cells. ( E ) DNA recognition sequence of SMC1A and RAD21 in WT or Asxl1 −/− LK cells. The SMC1A and RAD21 recognized identical DNA motif as CTCF.

Article Snippet: Genomic DNA regions of interest were isolated using antibodies against ASXL1 (Santa Cruz Biotechnology, sc-85283), SMC1A (Abcam, ab133643), and RAD21 (Abcam, ab154769).

Techniques: ChIP-sequencing, Binding Assay, Sequencing

Journal: Science Advances

Article Title: ASXL1 interacts with the cohesin complex to maintain chromatid separation and gene expression for normal hematopoiesis

doi: 10.1126/sciadv.1601602

Figure Lengend Snippet: ( A ) The heatmap shows the differentially expressed genes associated with loci of no changes in SMC1A and RAD21 occupancy in Asxl1 −/− BM LK cells. ( B ) The heatmap shows the differentially expressed genes associated with loss of SMC1A and/or RAD21 in Asxl1 −/− BM LK cells. ( C ) The GO analysis of the 237 up-regulated genes (of the ~1600 genes with loss of RAD21 and/or SMC1A occupancy in Asxl1 −/− LK cells) in Asxl1 −/− LK cells compared to the WT LK cells. ( D ) GO analysis of the 65 down-regulated genes (of ~1600 genes with loss of RAD21 and/or SMC1A occupancy in Asxl1 −/− LK cells) in Asxl1 −/− LK cells compared to the WT LK cells. The P values of each GO term are represented by the red dots, and the gene set counts are represented by the bars. ( E ) Heatmap of differentially expressed genes of myeloid malignancy relevance within Asxl1/SMC1/RAD21 overlapping loci in Asxl1 −/− LK cells. ( F and G ) Genome browser tracks of the Cbfb and Fus locus with overlapping ASXL1, SMC1A, or RAD21 peaks. ( H ) Relative RNA level of Cbfb, Fus, and Stat3 in LK cells as determined by qPCR. Data are represented as means ± SEM from three independent experiments. *** P < 0.001 and ** P < 0.01.

Article Snippet: Genomic DNA regions of interest were isolated using antibodies against ASXL1 (Santa Cruz Biotechnology, sc-85283), SMC1A (Abcam, ab133643), and RAD21 (Abcam, ab154769).

Techniques:

Journal: Nature immunology

Article Title: Smc3 dosage regulates B cell transit through germinal centers and restricts their malignant transformation

doi: 10.1038/s41590-020-00827-8

Figure Lengend Snippet: (a) Box plots showing transcripts per million (TPM) of cohesin core subunits ( Smc3 , Smc1a , Stag1 , Stag2 and Rad21 ) and regulator proteins ( Nipbl , Wapl , Pds5a , Pds5b ) in mouse and human (b) . NB: naive B cells, CB: centroblasts, CC: centrocytes, PC: splenic plasma cells, TPC: tonsillar plasma cells, BMPC: bone marrow plasma cells. (c) Scheme depicting Smc3 conditional knockout model breeding strategy and (d) genotyping strategy. (e) Gating strategy used for flow cytometric analysis of GC B cells. (f) Genotyping of sorted GC B cells. Representative gel of one experiment out of four performed. (g) Representative flow cytometry plots showing total B cells (B220 + cells) and follicular B cells (B220 + CD23 hi CD21 lo cells) and marginal zone B cell populations (B220 + CD23 lo CD21 hi cells) and quantitative data for one representative experiment out of 4 performed in Smc3 wt/wt (n=6), Smc3 wt/– (n=4) and Smc3 –/– (n=3) as shown (right). One-way ANoVA followed by Tukey test for multiple comparisons was used. (h-i) Quantification of GC B cells, defined as B220 + Fas + CD38 – , 4 days (h , p=0.002) or 15 days (I , p=0.0434) after SRBC immunization. Two-tail unpaired t -test was used. (j) RT-qPCR comparing Smc3 mRNA expression in GC B cells (centroblasts, CB and centrocytes, CC), plasmablasts (PB) and plasma cells (PC) in Cγ1 wt/cre ; Smc3 wt/wt and Cγ1 wt/cre ; Smc3 wt/– mice as indicated. Two-tail unpaired t -test was used. CB, p=0.0039, CC: p=0.0005, PB: p=0.0041 and PC: p=0.0271. (k) Capillary immunoblot analyses comparing Smc3, Smc1a, Rad21 and Sag2 protein levels in centroblasts (CB), centrocytes (CC) and naïve B cells (NBC) from Cγ1 wt/cre ; Smc3 wt/wt (n=3) and Cγ1 wt/cre ; Smc3 wt/– (n=3) mice as indicated. Representative gel our of two experiments performed. Right panels show quantifications of the cohesin complex subunits using β-actin protein as loading control. Two-way ANoVA was used. **p<0.01. In (a) and (b) box plots show the median as center, first and third quartiles as the box hinges, and whiskers extend to the smallest and largest value no further than the 1.5 × interquartile range (IQR) away from the hinges. In (g) , (h) , (i), (j) and (k) data are presented as mean +/− SD.

Article Snippet: Protein lysates were resolved using the fully automated capillary immnoblot Jess instrument (ProteinSimple), and probed with primary antibodies against Smc3, Smc1a, Rad21 (Cell Signaling) or β-actin (Sigma Aldrich).

Techniques: Clinical Proteomics, Knock-Out, Flow Cytometry, Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: Architectural proteins CTCF and cohesin have distinct roles in modulating the higher order structure and expression of the CFTR locus

doi: 10.1093/nar/gku648

Figure Lengend Snippet: CTCF and cohesin binding are interdependent at the CFTR locus. ( A ) UCSC Genome Browser graphic of the CFTR locus showing CTCF/cohesin binding sites (ENCODE data) marked by vertical black bars. ( B ) and ( C ) ChIP for CTCF, RAD21 and SMC1 in Caco2 cells transfected with negative control (NC) siRNA (gray) or siRNA targeting CTCF (B) or RAD21 (C) (black) for knockdown (KD). Data from one representative experiment are shown as percent recovery over input, n ≥ 2. qPCR was performed in duplicate at each site and error bars are ± SEM. Arrows denote sites of particular interest discussed in the text. Inset panels show relative siRNA-mediated depletion of CTCF or RAD21.

Article Snippet: Standard protocols were used and protein levels assayed with antibodies against CFTR (570, a generous gift from T Jensen and JR Riordan, ( )), CTCF (Millipore 07–729), RAD21 (Millipore 05–908) and β-tubulin (Sigma–Aldrich T4026).

Techniques: Binding Assay, Transfection, Negative Control

Journal: Nucleic Acids Research

Article Title: Architectural proteins CTCF and cohesin have distinct roles in modulating the higher order structure and expression of the CFTR locus

doi: 10.1093/nar/gku648

Figure Lengend Snippet: Depletion of CTCF or RAD21 alters long-range interactions across the CFTR locus. ( A )–( D ) q3C interactions in cells treated with NC siRNA (gray) or siRNA targeting CTCF (A) and (B) (black), or RAD21 (C) and (D) (black). The 3C bait (dotted line) is in a HindIII fragment at the CFTR promoter (A) and (C) or at the −20.9 kb insulator element (B) and (D). ( E ) ChIP for FOXA2 on cells transfected with NC (gray) or FOXA1/2 siRNA (black). Data are shown as percent recovery over input. ( F ) q3C interactions in NC (gray) or FOXA1/2 siRNA treated cells (black). The 3C bait (dotted line) is in a HindIII fragment at the CFTR promoter. Multiple sites were tested for interactions within distal HindIII fragments across the CFTR locus. x -axis = position relative to translational start site of CFTR , y -axis = interaction frequency relative to that between two fragments in the ubiquitously expressed ERCC3 gene. Data shown are from one representative q3C experiment, n = 3. Error bars represent the ± SEM of duplicate qPCRs for each fragment. ∧ indicate sites that are statistically different after knockdown as determined by an unpaired, two-tailed Student's t -test, n = 3, P -values listed in Supplementary Table S2. Other sites of interest marked with arrows. Inset panels show relative siRNA-mediated depletion of CTCF or RAD21.

Article Snippet: Standard protocols were used and protein levels assayed with antibodies against CFTR (570, a generous gift from T Jensen and JR Riordan, ( )), CTCF (Millipore 07–729), RAD21 (Millipore 05–908) and β-tubulin (Sigma–Aldrich T4026).

Techniques: Transfection, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Architectural proteins CTCF and cohesin have distinct roles in modulating the higher order structure and expression of the CFTR locus

doi: 10.1093/nar/gku648

Figure Lengend Snippet: Altered long-range interactions after CTCF and RAD21 depletion. ( A ) and ( B ) q3C interactions in cells treated with NC siRNA (gray) or siRNAs targeting CTCF and RAD21 (black), n = 3. The 3C bait (dotted line) is either at the CFTR promoter (A) or the −20.9 kb site (B). ∧ indicate sites that are statistically different after knockdown as determined by an unpaired, two-tailed Student's t -test, P -values listed in Supplementary Table S2. Other sites of interest are marked with arrows. Experimental details are in Figure legend.

Article Snippet: Standard protocols were used and protein levels assayed with antibodies against CFTR (570, a generous gift from T Jensen and JR Riordan, ( )), CTCF (Millipore 07–729), RAD21 (Millipore 05–908) and β-tubulin (Sigma–Aldrich T4026).

Techniques: Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Architectural proteins CTCF and cohesin have distinct roles in modulating the higher order structure and expression of the CFTR locus

doi: 10.1093/nar/gku648

Figure Lengend Snippet: siRNA-mediated depletion of architectural proteins augments CFTR expression. ( A ), ( D ) and ( G ) Western blots show siRNA-mediated depletion of CTCF (A), RAD21 (D), or both (G) in Caco2 cells with β-tubulin as the loading control. ( B ) and ( E ) ImageJ quantification of western blots shown in (A) and (D), respectively. ( C ), ( F ) and ( H ) RT-qPCR for CFTR expression in Caco2 cells after knockdown of CTCF (C), RAD21 (F) or both (H) (black) compared to NC siRNA treated cells (gray). ( I )–( K ) Western blots show CFTR levels after loss of CTCF (I), RAD21 (J) or both factors (K) in Caco2 cells with β-tubulin as the loading control. ( L ) ImageJ quantification of the CFTR western blots, n ≥ 3. * P < 0.05; ** P < 0.01 as determined by an unpaired, two-tailed Student's t -test. CT/RA KD is short for CTCF and RAD21 knockdown.

Article Snippet: Standard protocols were used and protein levels assayed with antibodies against CFTR (570, a generous gift from T Jensen and JR Riordan, ( )), CTCF (Millipore 07–729), RAD21 (Millipore 05–908) and β-tubulin (Sigma–Aldrich T4026).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Architectural proteins CTCF and cohesin have distinct roles in modulating the higher order structure and expression of the CFTR locus

doi: 10.1093/nar/gku648

Figure Lengend Snippet: Depletion of architectural proteins alters histone modifications and transcription factor occupancy. ( A )–( C ) ChIP for H3K9me3 (A), H3K9ac (B) and H3K27ac (C) in Caco2 cells transfected with NC siRNA (gray) or a siRNA targeting CTCF (black). Data are shown as percent recovery over input and each histone modification is normalized to recovery over input of unmodified histone H3 at each site, n = 3. ∧ indicate sites that are statistically different after knockdown as determined by an unpaired, two-tailed Student's t -test, P -values are listed in Supplementary Table S2. Other sites of interest are marked with arrows. ( D )–( F ) ChIP for FOXA1 (D), FOXA2 (E) and CDX2 (F) in Caco2 cells transfected with NC siRNA (gray) or siRNAs targeting CTCF and RAD21 (black). Data are shown as percent recovery over input, n = 3. * P < 0.05; ** P < 0.01 as determined by an unpaired, two-tailed Student's t -test.

Article Snippet: Standard protocols were used and protein levels assayed with antibodies against CFTR (570, a generous gift from T Jensen and JR Riordan, ( )), CTCF (Millipore 07–729), RAD21 (Millipore 05–908) and β-tubulin (Sigma–Aldrich T4026).

Techniques: Transfection, Modification, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Architectural proteins CTCF and cohesin have distinct roles in modulating the higher order structure and expression of the CFTR locus

doi: 10.1093/nar/gku648

Figure Lengend Snippet: Altered nuclear positioning of CFTR alleles after loss of CTCF or RAD21. ( A )–( C ) Representative FISH images for CFTR in Caco2 cells treated with NC siRNA (A), CTCF siRNA (B), or RAD21 siRNA (C). Nuclei are stained with DAPI and the CFTR alleles are white dots. ( D ) Quantification of the minimum distance to the nuclear periphery of the CFTR alleles in all three conditions. ** P < 0.01 as determined by an unpaired, two-tailed Student's t -test.

Article Snippet: Standard protocols were used and protein levels assayed with antibodies against CFTR (570, a generous gift from T Jensen and JR Riordan, ( )), CTCF (Millipore 07–729), RAD21 (Millipore 05–908) and β-tubulin (Sigma–Aldrich T4026).

Techniques: Staining, Two Tailed Test