Journal: International Journal of Cell Biology

Article Title: Nesprin-2 Interacts with Condensin Component SMC2

doi: 10.1155/2017/8607532

Figure Lengend Snippet: Characterization of the SMC domain of Nesprin-2. (a) Schematic of Nesprin-2 (not drawn to scale). The location of the SMC domain (spectrin repeats 11–13) and the C-terminal spectrin repeats (53–56) is shown. Epitopes of antibodies used are indicated above the schematic. ABD, actin binding domain; ovals, spectrin repeats. The spectrin repeat domain starts at position 308. (b) Sequence comparison of the Nesprin-2-SMC domain with coiled-coil regions of SMC2 and SMC4. The sequence comparison was performed using LALIGN, the Pairwise Sequence Alignment tool from EMBL-EBI ( https://www.ebi.ac.uk/Tools/psa/lalign/ ). Nesprin-2 (NCBI GenBank accession number AF435011.1), SMC2 (NCBI GenBank accession number O95347.2), and SMC4 (NCBI GenBank accession number Q8WXH0.3) were used. :, identical amino acid; ., conservative substitution. (c) Analysis of Nesprin-2-SMC by gel filtration chromatography. UV traces of the elution profile are shown. Nesprin-2 SMC (calculated molecular weight 39 kDa). Molecular weight markers were ovalbumin (43 kDa), conalbumin (75 kDa), and aldolase (158 kDa). (d) Analysis of chemically crosslinked Nesprin-2-SMC. Zero-length cross-linking reagent EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) was used at decreasing concentrations. The proteins were separated by SDS-PAGE (10% acrylamide) and stained with Coomassie Blue. (e) Schematic representation of Myc-tagged Nesprin-2-SMC polypeptides. Amino acid positions refer to human Nesprin-2 giant (accession number AF435011.1). (f) Interaction of GST-Nesprin-2-SMC with individual Myc-tagged spectrin repeats derived from Nesprin-2-SMC and expressed in COS7 cells. GST-Nesprin-2-SMC was used for pulldown (right panel). Western blots were probed with mAb 9E10 specific for Myc. Asterisk, endogenous Myc . (g) Specificity of the Nesprin-2-SMC interaction. Myc-SR53–56 expressed in COS7 cells was used for pulldowns with GST for control and GST-Nesprin-2-SMC. COS7 and COS7 Myc-SR53–56 represent whole cell lysates. The Ponceau S stained blot and the corresponding blot probed with mAb 9E10 are shown. MW, molecular weight marker (from top to bottom: 200, 130, 100, 70, 55, 35, and 25 kDa).

Article Snippet: The following antibodies were used: mouse monoclonal anti-Nesprin-2 mAb K20-478 raised against the actin binding domain (ABD) of Nesprin-2 (residues 1–285) [ ] (IF, 1 : 200; hybridoma supernatant, WB, 1 : 10), rabbit polyclonal antibodies pAbK1 raised against spectrin repeats in the C-terminal region of Nesprin-2 [ ] (IF, 1 : 100; WB, 1 : 1,000), Nesprin-1 specific mAb K43-322-2 raised against N-terminal spectrin repeats 10 and 11 of Nesprin-1 [ ] (hybridoma supernatant, undiluted), GFP-specific mAb K3-184-2 [ ] (hybridoma supernatant, IF, 1 : 2; WB, 1 : 10), Myc-specific mAb 9E10 [ ] (hybridoma supernatant, IF, undiluted; WB, 1 : 10), pAb against GST [ ] (WB, 1 : 50,000), mAb K84-913 against GST (hybridoma supernatant, WB 1 : 10), pAb Lamin B1 (Abcam ab16048, IF, 1 : 200; WB, 1 : 4,000), pAb SMC2 (Novus Biologicals NB100-373, IF, 1 : 100; WB, 1 : 2,000), WB: mAb SMC4 (Abcam ab179803 1 : 2,000), IF: pAb SMC4 (Abcam ab17958, 1 : 500), pAb SMC1 (Abcam ab21583, WB 1 : 1000), goat SMC3 (Santa Cruz Biotechnology, sc-8135, WB 1 : 50), rabbit CAP-H (Biomol-Bethyl A300-603A-T, WB 1 : 1000), pAb CAP-H2 (Biomol-Bethyl A302-275A, WB 1 : 4000), mAb PDI (Abcam ab2792, 1 : 100), pAb calreticulin (Thermo Fisher PA3-900, IF 1 : 50–200), and rat mAb YL1/2 specific for α -tubulin (1 : 5). mAb K81-116-6 (hybridoma supernatant, undiluted) directed against the SMC domain in Nesprin-2 was generated in this study.

Techniques: Binding Assay, Sequencing, Comparison, Filtration, Chromatography, Molecular Weight, SDS Page, Staining, Derivative Assay, Western Blot, Control, Marker

Journal: International Journal of Cell Biology

Article Title: Nesprin-2 Interacts with Condensin Component SMC2

doi: 10.1155/2017/8607532

Figure Lengend Snippet: Interaction of Nesprin-2-SMC and Nesprin-2 with SMC2 and SMC4. (a) Precipitation of SMC2 and SMC4 with GST-Nesprin-2-SMC from HaCaT cell lysates. Precipitates were resolved on SDS-polyacrylamide gels (10% acrylamide) and probed with SMC2 and SMC4 specific antibodies. SPN, supernatant after pulldown; PD, pulldown. The lower molecular weight band in the SMC2 pulldown is presumably a breakdown product. (b) Immunoprecipitation of SMC2 from HaCaT cell lysates with Nesprin-2 specific mAbK20-478 and of Nesprin-2 with SMC2 specific antibodies. GFP-specific monoclonal antibodies were used for control. The antibodies used for immunoprecipitation are indicated above the panels (IP). The blots were probed with the antibodies listed on the right (WB). Immunoprecipitates were resolved on gradient gels (3–12% acrylamide) and 10% acrylamide gels as appropriate. The data are from one blot; however, the input was not directly adjacent to the SMC2 IP. (c) Interaction of CAP-H2 (condensin II) and CAP-H (condensin I) with Nesprin-2-SMC. Pulldowns were performed with HaCaT cell lysates and GST for control and GST-Nesprin-2-SMC as indicated. Unsynchronized cells were used for the experiments shown in (a)–(c). (d) Analysis of the Nesprin-2-SMC interaction with SMC2 during the cell cycle. HaCaT cells were synchronized with RO-3306 or other reagents as described in Materials and Methods in order to obtain the relevant cell cycle phases. Cell cycle phases were assessed by FACS analysis; the results are depicted in the accompanying diagram. Pulldown was carried out with GST-Nesprin-2-SMC bound to GST-Sepharose. GST was used for control. The blot was probed with SMC2 specific antibodies. (e) Localization of Nesprin-2 as detected with mAb K81-116-6 (green) during mitosis in HaCaT cells. DNA was stained with DAPI. Arrow points to filamentous staining across the chromosomes. (f) Nesprin-2 distribution in HaCaT cells during mitosis as detected with mAb K20-478 (green) and pAbK1 (red). DNA was detected with DAPI. Bar, 10 μ m. (g) Nesprin-2 presence on chromosomes. Different Z-stacks (from top to bottom: 0 μ m, 0.21 μ m 0.42 μ m, and 0.84 μ m) from a COS7 cell in anaphase stained with mAb K20-478. DNA was stained with DAPI. Bar, 5 μ m.

Article Snippet: The following antibodies were used: mouse monoclonal anti-Nesprin-2 mAb K20-478 raised against the actin binding domain (ABD) of Nesprin-2 (residues 1–285) [ ] (IF, 1 : 200; hybridoma supernatant, WB, 1 : 10), rabbit polyclonal antibodies pAbK1 raised against spectrin repeats in the C-terminal region of Nesprin-2 [ ] (IF, 1 : 100; WB, 1 : 1,000), Nesprin-1 specific mAb K43-322-2 raised against N-terminal spectrin repeats 10 and 11 of Nesprin-1 [ ] (hybridoma supernatant, undiluted), GFP-specific mAb K3-184-2 [ ] (hybridoma supernatant, IF, 1 : 2; WB, 1 : 10), Myc-specific mAb 9E10 [ ] (hybridoma supernatant, IF, undiluted; WB, 1 : 10), pAb against GST [ ] (WB, 1 : 50,000), mAb K84-913 against GST (hybridoma supernatant, WB 1 : 10), pAb Lamin B1 (Abcam ab16048, IF, 1 : 200; WB, 1 : 4,000), pAb SMC2 (Novus Biologicals NB100-373, IF, 1 : 100; WB, 1 : 2,000), WB: mAb SMC4 (Abcam ab179803 1 : 2,000), IF: pAb SMC4 (Abcam ab17958, 1 : 500), pAb SMC1 (Abcam ab21583, WB 1 : 1000), goat SMC3 (Santa Cruz Biotechnology, sc-8135, WB 1 : 50), rabbit CAP-H (Biomol-Bethyl A300-603A-T, WB 1 : 1000), pAb CAP-H2 (Biomol-Bethyl A302-275A, WB 1 : 4000), mAb PDI (Abcam ab2792, 1 : 100), pAb calreticulin (Thermo Fisher PA3-900, IF 1 : 50–200), and rat mAb YL1/2 specific for α -tubulin (1 : 5). mAb K81-116-6 (hybridoma supernatant, undiluted) directed against the SMC domain in Nesprin-2 was generated in this study.

Techniques: Molecular Weight, Immunoprecipitation, Bioprocessing, Control, Staining

Journal: International Journal of Cell Biology

Article Title: Nesprin-2 Interacts with Condensin Component SMC2

doi: 10.1155/2017/8607532

Figure Lengend Snippet: SMC2 (a) and SMC4 (b) in HaCaT keratinocytes treated with control shRNA (upper panels) and treated with Nesprin-2-SMC domain specific shRNA (lower panels). Nesprin-2 was detected with mAb K20-478. Bar, 10 μ m. (c) Localization of Nesprin-2 after siRNA mediated knockdown of SMC2 in COS7 cells. Staining was with SMC2 specific antibodies and mAb K20-478 for Nesprin-2. Bar, 5 μ m. (d) Evaluation of the SMC2 knockdown. SMC2 fluorescence intensity was measured in the center of mitotic chromosomes. 10 siRNA treated cells and 12 control cells (control treatment) were analyzed ( ∗∗∗ P value = 0.0001).

Article Snippet: The following antibodies were used: mouse monoclonal anti-Nesprin-2 mAb K20-478 raised against the actin binding domain (ABD) of Nesprin-2 (residues 1–285) [ ] (IF, 1 : 200; hybridoma supernatant, WB, 1 : 10), rabbit polyclonal antibodies pAbK1 raised against spectrin repeats in the C-terminal region of Nesprin-2 [ ] (IF, 1 : 100; WB, 1 : 1,000), Nesprin-1 specific mAb K43-322-2 raised against N-terminal spectrin repeats 10 and 11 of Nesprin-1 [ ] (hybridoma supernatant, undiluted), GFP-specific mAb K3-184-2 [ ] (hybridoma supernatant, IF, 1 : 2; WB, 1 : 10), Myc-specific mAb 9E10 [ ] (hybridoma supernatant, IF, undiluted; WB, 1 : 10), pAb against GST [ ] (WB, 1 : 50,000), mAb K84-913 against GST (hybridoma supernatant, WB 1 : 10), pAb Lamin B1 (Abcam ab16048, IF, 1 : 200; WB, 1 : 4,000), pAb SMC2 (Novus Biologicals NB100-373, IF, 1 : 100; WB, 1 : 2,000), WB: mAb SMC4 (Abcam ab179803 1 : 2,000), IF: pAb SMC4 (Abcam ab17958, 1 : 500), pAb SMC1 (Abcam ab21583, WB 1 : 1000), goat SMC3 (Santa Cruz Biotechnology, sc-8135, WB 1 : 50), rabbit CAP-H (Biomol-Bethyl A300-603A-T, WB 1 : 1000), pAb CAP-H2 (Biomol-Bethyl A302-275A, WB 1 : 4000), mAb PDI (Abcam ab2792, 1 : 100), pAb calreticulin (Thermo Fisher PA3-900, IF 1 : 50–200), and rat mAb YL1/2 specific for α -tubulin (1 : 5). mAb K81-116-6 (hybridoma supernatant, undiluted) directed against the SMC domain in Nesprin-2 was generated in this study.

Techniques: Control, shRNA, Knockdown, Staining, Fluorescence

Journal: bioRxiv

Article Title: Extensive mutual influences of SMC complexes shape 3D genome folding

doi: 10.1101/2024.07.31.606012

Figure Lengend Snippet: a , Schematic showing the edited Wapl locus and genotyping strategy. b , Genotyping results of positive clones targeting the Wapl locus. c , Schematic showing the edited Smc2 locus and genotyping strategy. d , Genotyping results of positive clones targeting the Smc2 locus. e , Flow cytometry plots showing progressive loss of Wapl or/and SMC2 fluorescence signals upon dTag13 or/and 5-Ph-IAA treatment. The parental cell line was shown as negative control. f , Western blots showing degradation of Wapl or/and SMC2 upon dTag13 or/and 5-Ph-IAA treatment. g , Immunofluorescence image showing mitotic retention of Rad21 upon Wapl depletion. Scale bar: 5μm. h , Quantification of the percentage of mitotic Rad21 retention upon Wapl loss (n=3 for each clone). Error bar denotes SEM. P values were calculated using two-sided student’s t -test. i , Metaphase spread analysis showing “closed-arm” mitotic cells in the absence of Wapl, indicating mitotic retention of cohesin. Scale bar: 5μm. P values were calculated using two-sided student’s t -test. j , Representative image showing the presence of lagging chromosomes in ana/telophase upon Wapl depletion. Scale bar: 5μm. Two independent clones were shown. k , Quantification of lagging chromosomes in Wapl-replete and depleted mitotic cells (n=3 for each clone). Error bar denotes SEM. P values were calculated using two-sided student’s t -test. l , Line graph showing cell growth defects in Wapl depleted, SMC2 depleted or Wapl and SMC2 co-depleted cells. Error bar denotes SEM. P values were calculated using two-sided student’s t -test.

Article Snippet: Blots were probed with antibodies against SMC2 (Abclonal, A17867, 1:1000), Wapl (CST, 77428, 1:1000) and β-actin (ABclonal, AC004; 1:5,000) and imaged using a BioRad ChemiDoc MP Imaging System.

Techniques: Clone Assay, Flow Cytometry, Fluorescence, Negative Control, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: Extensive mutual influences of SMC complexes shape 3D genome folding

doi: 10.1101/2024.07.31.606012

Figure Lengend Snippet: a , Experimental design, showing the strategy of prometaphase arrest in combination with 5-Ph-IAA or/and dTag13 treatment. b , Flow cytometry plot showing the gating strategy to purify Wapl-depleted and Wapl and SMC2 co-depleted mitotic cells. c , Representative images showing FACS purified Wapl-depleted or Wapl and SMC2 co-depleted mitotic cells. Two independent experiments were performed. Scale bar: 20μm.

Article Snippet: Blots were probed with antibodies against SMC2 (Abclonal, A17867, 1:1000), Wapl (CST, 77428, 1:1000) and β-actin (ABclonal, AC004; 1:5,000) and imaged using a BioRad ChemiDoc MP Imaging System.

Techniques: Flow Cytometry, Purification

Journal: bioRxiv

Article Title: Extensive mutual influences of SMC complexes shape 3D genome folding

doi: 10.1101/2024.07.31.606012

Figure Lengend Snippet: a , Venn diagram showing the intersection results of Rad21 peaks in the Wapl deficient G1-phase samples and Wapl and SMC2 co-depleted mitotic samples. b , Density heatmap plots showing the ChIP-seq signals of Rad21 peaks that were lost, maintained or gained during mitosis. c , Meta region plots showing the corresponding mitotic CTCF ChIP-seq signals for Rad21 peaks that were lost, maintained or gained. Note that compared to the lost ones, the maintained Rad21 peaks were co-localized by stronger mitotic CTCF binding. CTCF ChIP-seq data was adapted from our previous study . d , Dot plots showing the average Rad21 ChIP-seq signals for peaks that were lost, maintained or gained during mitosis (n=4 and 5 for G1-phase and mitotic samples respectively). P values were calculated using two-sided student’s t -test. e , De-novo motif enrichment analysis for the mitotic specific Rad21 peaks. f , Box plot showing that mitotic specific Rad21 peaks were significantly larger compared to the interphase Rad21 peaks. For both box plots, central lines denote medians; box limits denote 25th–75th percentile; whiskers denote 5th–95th percentile. P values were calculated using a two-sided Wilcoxon signed-rank test. g , Meta region plots showing that mitotic specific Rad21 peaks were heavily decorated with active histone marks. h , Pie charts showing that the mitotic specific Rad21 peaks were significantly more likely to co-localize with CREs.

Article Snippet: Blots were probed with antibodies against SMC2 (Abclonal, A17867, 1:1000), Wapl (CST, 77428, 1:1000) and β-actin (ABclonal, AC004; 1:5,000) and imaged using a BioRad ChemiDoc MP Imaging System.

Techniques: ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Extensive mutual influences of SMC complexes shape 3D genome folding

doi: 10.1101/2024.07.31.606012

Figure Lengend Snippet: a , Schematic showing the genome editing strategy to generate the Wapl dTag /Sororin mAID cell line. b , Metaphase spread analysis showing a complete loss of sister-chromatid cohesion in Sororin depleted mitotic Wapl dTag / Sororin mAID cells. Co-depletion of Wapl and Sororin, abrogated this phenotype. Scale bar: 10μm. c , Quantification of ( b ) showing the percentage of sister-chromatid dissociation in indicated samples. P values were calculated by two-sided student’s t -test. d , Upper panel: G2/M arrest/release strategy to obtain condensin-replete mitotic chromosomes with extrusive-cohesin (“heavy load”). For simplicity, we employed three illustrative symbols to represent condensin, extrusive-cohesin and cohesive-cohesin. These symbols are used hereafter throughout the study. Lower panel: G2/M arrest/release strategy to obtain condensin-deficient mitotic chromosomes with extrusive-cohesin (“heavy load”). e , Schematic showing the genome editing strategy to generate the Wapl dTag /SMC2 mAID /Sororin mAID cell line. f , Venn-diagrams showing the loss of extrusive-cohesin peaks (“heavy load”) in the presence of condensin. Peaks co-localized to CTCF, CREs or both were shown separately. g , Density heatmap plots showing reduced mitotic extrusive-cohesin peak strength (“heavy load”) in the presence of condensin. Peaks co-localized to CTCF, CREs or both were plotted separately. h , Browser tracks showing that condensin disrupts extrusive-cohesin (Rad21) peaks (“heavy load”). Peaks coincided with CTCF and CRE were indicated by blue and red arrows respectively. Tracks of CTCF and H3K27ac from parent cells were shown. i , Similar to ( d ) showing the strategies to obtain condensin-replete or deficient mitotic chromosomes with low levels of extrusive-cohesin (“light load”). ( j - l ) Similar to ( f - h ) with Venn-diagrams ( j ), Density heatmap plots ( k ) and browser tracks ( l ) showing the complete disruption of extrusive-cohesin (“light load”) by condensin.

Article Snippet: Blots were probed with antibodies against SMC2 (Abclonal, A17867, 1:1000), Wapl (CST, 77428, 1:1000) and β-actin (ABclonal, AC004; 1:5,000) and imaged using a BioRad ChemiDoc MP Imaging System.

Techniques: Disruption

Journal: bioRxiv

Article Title: Extensive mutual influences of SMC complexes shape 3D genome folding

doi: 10.1101/2024.07.31.606012

Figure Lengend Snippet: a , Schematic showing the edited Cdca5 (encoding Sororin), Wapl and Smc2 loci and the respective genotyping strategy. b , Genotyping results of the Wapl dTag /Sororin mAID clones. c , Fluorescence images showing loss of Wapl and Sororin fluorescence signals upon 4 hours of dTag13 and 5-Ph-IAA treatment in the Wapl dTag / Sororin mAID cells. d , Schematic showing the strategy to determine G2/M arrest length. e , Metaphase spread analysis showing a more extensive dissociation of sister-chromatids in the Wapl dTag / Sororin mAID cells after 16 hours compared to 6 hours of RO-3306 mediated G2 arrest. f , Quantification of ( d & e ), showing progressive dissociation of sister-chromatids as G2-arrest duration were extended. Longer G2-arrest allowed more complete removal of cohesive-cohesin by Wapl. P values were calculated by two-sided student’s t -test. g , Genotyping results of Wapl dTag /SMC2 mAID /Sororin mAID clones. h , Fluorescence images showing loss of Wapl, Sororin and SMC2 fluorescence signals upon 4 hours of dTag13 and 5-Ph-IAA treatment in the Wapl dTag /SMC2 mAID /Sororin mAID cells.

Article Snippet: Blots were probed with antibodies against SMC2 (Abclonal, A17867, 1:1000), Wapl (CST, 77428, 1:1000) and β-actin (ABclonal, AC004; 1:5,000) and imaged using a BioRad ChemiDoc MP Imaging System.

Techniques: Clone Assay, Fluorescence

Journal: bioRxiv

Article Title: Extensive mutual influences of SMC complexes shape 3D genome folding

doi: 10.1101/2024.07.31.606012

Figure Lengend Snippet: a , Bar graph showing the number of loops identified by HICCUPS in interphase and mitotic samples. The fraction of structural and CRE loops in each sample were also shown. b , APA plots showing the composite structural loop signals in interphase and mitotic samples with eight distinct SMC protein complex configurations. Note that structural loops were appreciable in condensin-deficient mitotic cells loaded with extrusive-cohesin (green arrows). c , KR-balanced Hi-C contact maps showing the weakening of structural loops (green arrow) upon the introduction of cohesive-cohesin during mitosis. Maps for Wapl dTag /SMC2 mAID cells which expresses endogenous wildtype Sororin were shown on the right. Bin size: 25kb. d , Heatmap showing the clustering result structural loops based on their responses to cohesive-cohesin. e , APA plots for each cluster in ( d ) showing loss of structural loop signal intensity in the presence of cohesive-cohesin. Plot for Wapl dTag /SMC2 mAID cells which expresses endogenous wildtype Sororin were shown on the right. f, Meta-region plots showing Rad21 ChIP-seq signal strength at structural loop anchors from each cluster. g , Box plots showing the quantification of ( f ). For both box plots, central lines denote medians; box limits denote 25th–75th percentile; whiskers denote 5th–95th percentile. P values were calculated using a two-sided Wilcoxon signed-rank test. h , Box plots showing the sizes of structural loops from cluster1, cluster2, cluster3 and cluster4. For both box plots, central lines denote medians; box limits denote 25th–75th percentile; whiskers denote 5th–95th percentile. P values were calculated using a two-sided Wilcoxon signed-rank test.

Article Snippet: Blots were probed with antibodies against SMC2 (Abclonal, A17867, 1:1000), Wapl (CST, 77428, 1:1000) and β-actin (ABclonal, AC004; 1:5,000) and imaged using a BioRad ChemiDoc MP Imaging System.

Techniques: Hi-C, ChIP-sequencing

Journal: Cell Cycle

Article Title: Inactivation of SMC2 shows a synergistic lethal response in MYCN -amplified neuroblastoma cells

doi: 10.4161/cc.27983

Figure Lengend Snippet: Figure 1. Smc2 expression in neuroblastoma model mice and SMC2 expression in human neuroblastoma cell lines. ( A ) Results of a microarray analysis of the relative expression levels of Smc2 in ganglia of wt mice (lanes 1 and 2), and precancerous (lanes 3 and 4) and tumor lesions (lanes 5 and 6) of homozygous MYCN Tg mice. ( B ) Semi-quantitative (left) and quantitative (right) RT-PCR analyses of Smc2 and Gapdh (control) expression levels in 3 precancerous lesion samples from hemizygous MYCN Tg mice (hemi) and ganglia of wt mouse. ( C ) Semi-quantitative (left) and quantitative (right) RT-PCR analyses of human SMC2 expression levels in various human neuroblastoma cell lines. SH-SY5Y, SK-N-AS, and SH-EP cells have a single copy of MYCN, and IMR32 and SK-N-BE(2) have amplified MYCN . The expression levels of Smc2 and SMC2 detected by RT-qPCR were normalized to those of Gapdh and GAPDH respectively.

Article Snippet: The following antibodies were used: anti-MYCN monoclonal antibodies (OP13, Calbiochem and NB200-109, Novus Biologicals); anti-MYCN monoclonal antibody (B8.4.B) (sc-53993, Santa Cruz); anti-SMC2 rabbit polyclonal antibodies (GTX10411, GeneTex; and ab10412, Abcam); anti-β-ACTIN monoclonal antibody (AC-15) (A5441, Sigma); anti-SMC4 rabbit polyclonal antibody (ab17958, Abcam); anti-CAP-D2 rabbit polyclonal antibody (A300-601A, Bethyl Laboratories); and anti-phospho-histone H2A.X (Ser139) mouse monoclonal antibody (JBW301, Millipore).

Techniques: Expressing, Microarray, Reverse Transcription Polymerase Chain Reaction, Control, Amplification, Quantitative RT-PCR

Journal: Cell Cycle

Article Title: Inactivation of SMC2 shows a synergistic lethal response in MYCN -amplified neuroblastoma cells

doi: 10.4161/cc.27983

Figure Lengend Snippet: Figure 2. Overexpression of MYCN induces SMC2 expression in human neuroblastoma cells. ( A and B ) A CMV-driven plasmid containing MYCN was introduced into the SH-EP MYCN single copy cell line. After live cell sorting, the levels of human SMC2 mRNA and protein were measured by semi-quantitative ( A , left) and quantitative ( A , right) RT-PCR, as well as by immunoblotting ( B ). The arrowhead in ( B ) indicates a non-specific band. The expression levels of SMC2 detected by RT-qPCR were normalized to those of GAPDH . ( C ) Schematic representation of the E-boxes identified in the human SMC2 gene. The light gray boxes indicate the 5′ and 3′ UTRs; the dark gray boxes indicate the exons; and the black circles represent putative E-boxes (MYCN-binding sites). The sequences of the E-boxes are shown. ( D ) ChIP followed by qPCR analysis of the E-boxes in the SMC2 gene was performed using control IgG or an anti-MYCN antibody in SH-EP cells expressing Venus (control) or MYCN. The 5-kb sequence upstream of the SMC2 gene was examined as a negative control. The data show the percentage of the target DNA precipitated with the control IgG or MYCN antibody and are represented as the mean ± SE of at least n = 3 independent experiments.

Article Snippet: The following antibodies were used: anti-MYCN monoclonal antibodies (OP13, Calbiochem and NB200-109, Novus Biologicals); anti-MYCN monoclonal antibody (B8.4.B) (sc-53993, Santa Cruz); anti-SMC2 rabbit polyclonal antibodies (GTX10411, GeneTex; and ab10412, Abcam); anti-β-ACTIN monoclonal antibody (AC-15) (A5441, Sigma); anti-SMC4 rabbit polyclonal antibody (ab17958, Abcam); anti-CAP-D2 rabbit polyclonal antibody (A300-601A, Bethyl Laboratories); and anti-phospho-histone H2A.X (Ser139) mouse monoclonal antibody (JBW301, Millipore).

Techniques: Over Expression, Expressing, Plasmid Preparation, FACS, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Binding Assay, Control, Sequencing, Negative Control

Journal: Cell Cycle

Article Title: Inactivation of SMC2 shows a synergistic lethal response in MYCN -amplified neuroblastoma cells

doi: 10.4161/cc.27983

Figure Lengend Snippet: Figure 3A–C. Knockdown of SMC2 induces DNA damage and apoptosis. ( A ) Growth of MYCN-overexpressed SH-EP cells and control SH-EP cells infected with non-target shRNA or SMC2 -specific shRNAs. Counting started 3 d after infection. On each day, 3 plates were counted and averaged. Data are shown as a ratio of the number of cells at 3 d after transfection and are represented as the mean ± SD of n = 3 independent repeats. ( B ) SMC2 knockdown efficiency of ( A ). ( C ) TUNEL staining of apoptotic IMR32 cells at 6 d after infection (upper panel) and quantification of apoptotic IMR32 cells at the indicated time-points (lower panel). On each day, 3 plates were counted and averaged. ( D ) Immunofluorescence of γ-H2AX and DAPI staining of IMR32 ( MYCN -amplified), SK-N-BE(2) ( MYCN -amplified), SK-N-AS ( MYCN -single copy), and SH-EP ( MYCN -single copy) cells infected with non-target or SMC2 -specific shRNA. Images were captured 3 d after infection. ( E ) Quantification of the γ-H2AX-positive cells shown in ( D ). Data are represented as the mean ± SD of n = 3 independent repeats. Their homoscedasticities were checked by f test. Statistical significance was evaluated with a 2-tailed, unpaired t test. ( F ) The percentage viability of SH-EP or MYCN overexpressed SH-EP cells infected with non-target or SMC2 -specific shRNA and treated with cisplatin (left) or campthotecin (right). Data are represented as the mean ± SE of n = 3 independent experiments. Their homoscedasticities were checked by f test. Statistical significance was evaluated with a 2-tailed, unpaired t test.

Article Snippet: The following antibodies were used: anti-MYCN monoclonal antibodies (OP13, Calbiochem and NB200-109, Novus Biologicals); anti-MYCN monoclonal antibody (B8.4.B) (sc-53993, Santa Cruz); anti-SMC2 rabbit polyclonal antibodies (GTX10411, GeneTex; and ab10412, Abcam); anti-β-ACTIN monoclonal antibody (AC-15) (A5441, Sigma); anti-SMC4 rabbit polyclonal antibody (ab17958, Abcam); anti-CAP-D2 rabbit polyclonal antibody (A300-601A, Bethyl Laboratories); and anti-phospho-histone H2A.X (Ser139) mouse monoclonal antibody (JBW301, Millipore).

Techniques: Knockdown, Control, Infection, shRNA, Transfection, TUNEL Assay, Staining, Immunofluorescence, Amplification

Journal: Cell Cycle

Article Title: Inactivation of SMC2 shows a synergistic lethal response in MYCN -amplified neuroblastoma cells

doi: 10.4161/cc.27983

Figure Lengend Snippet: Figure 4A and B. SMC2 interacts with MYCN and transcriptionally regulates DDR genes. ( A ) RT-qPCR analysis of the relative expression levels of DDR genes in IMR32 cells ( MYCN -amplified, top-left panel) and SK-N-AS cells ( MYCN single copy, top-right panel) infected with non-targeting or SMC2 -specific shRNA. Expression levels in SMC2 -specific shRNA-infected cells were normalized to those in the control cells. Bottom panels are shown SMC2 knockdown efficiency in each cells. ( B ) RT-qPCR analysis of the relative expression levels of DDR genes in MYCN-overexpressed SH-EP cells and control SH-EP cells as similar in ( A ). ( C ) Pull-down assay showing that MYCN interacts with SMC2 and SMC4. CMV-driven MYCN, along with CMV-driven Halo-control, Halo-SMC2 or Halo-SMC4 and Halo-MYCN proteins were expressed in 293T cells. A pull-down assay was performed using a Halo-tag. The proteins were detected with the indicated antibodies. WCE (TMR), TMR Direct ligand stained Halo-tag proteins in whole cell extract. ( D ) SMC2 and MYCN bind to the E-box motif in the NBS1 gene. The left panel shows the results of a ChIP assay of the E-box in the NBS1 gene performed using an anti-MYCN antibody in cells expressing Venus (control) or MYCN. The right panel shows the results of a ChIP assay of the E-box in the NBS1 gene performed using an anti-SMC2 antibody in MYCN -amplified IMR32 cells. A region situated 14.5 kb upstream of the E-box was used as a negative control. Data show the percentages of the target DNA precipitated with the antibodies and are represented as the mean ± SE of at least n = 3 independent qPCR experiments. ( E ) The protein level of SMC4 and CAP-D2 when SMC2 is knockdown in MYCN expressing SH-EP ( MYCN single copy) cells and control Venus expressing SH-EP cells. β-ACTIN is used as loading control.

Article Snippet: The following antibodies were used: anti-MYCN monoclonal antibodies (OP13, Calbiochem and NB200-109, Novus Biologicals); anti-MYCN monoclonal antibody (B8.4.B) (sc-53993, Santa Cruz); anti-SMC2 rabbit polyclonal antibodies (GTX10411, GeneTex; and ab10412, Abcam); anti-β-ACTIN monoclonal antibody (AC-15) (A5441, Sigma); anti-SMC4 rabbit polyclonal antibody (ab17958, Abcam); anti-CAP-D2 rabbit polyclonal antibody (A300-601A, Bethyl Laboratories); and anti-phospho-histone H2A.X (Ser139) mouse monoclonal antibody (JBW301, Millipore).

Techniques: Quantitative RT-PCR, Expressing, Amplification, Infection, shRNA, Control, Knockdown, Pull Down Assay, Staining, Negative Control

Journal: Cell Cycle

Article Title: Inactivation of SMC2 shows a synergistic lethal response in MYCN -amplified neuroblastoma cells

doi: 10.4161/cc.27983

Figure Lengend Snippet: Table 2. List of induced or repressed GO categolies belonging to DDR, DNA repair, and cell cycle-related classes in SMC2 -knockdown Venus- or MYCN-expressing SH-EP cells. Non-target shRNA-infected cells were used as a control

Article Snippet: The following antibodies were used: anti-MYCN monoclonal antibodies (OP13, Calbiochem and NB200-109, Novus Biologicals); anti-MYCN monoclonal antibody (B8.4.B) (sc-53993, Santa Cruz); anti-SMC2 rabbit polyclonal antibodies (GTX10411, GeneTex; and ab10412, Abcam); anti-β-ACTIN monoclonal antibody (AC-15) (A5441, Sigma); anti-SMC4 rabbit polyclonal antibody (ab17958, Abcam); anti-CAP-D2 rabbit polyclonal antibody (A300-601A, Bethyl Laboratories); and anti-phospho-histone H2A.X (Ser139) mouse monoclonal antibody (JBW301, Millipore).

Techniques: Knockdown, shRNA, Gene Expression, Modification, Binding Assay, Activity Assay, Protein Binding, Transduction

Journal: Cell Cycle

Article Title: Inactivation of SMC2 shows a synergistic lethal response in MYCN -amplified neuroblastoma cells

doi: 10.4161/cc.27983

Figure Lengend Snippet: Figure 5. Clinical data showing the relationship between SMC2 expression and patient prognosis in the Wang cohort. ( A ) The effects of SMC2 expression on the overall survival (OS) and event-free survival (EFS) rates of patients bearing MYCN -amplified and non-amplified tumors. Within each of the 2 tumor subsets considered, those with expression levels of SMC2 greater than the median (blue or green line) were compared with the remainder of the tumors in the subset (red or purple line) using a Kaplan–Meier analysis. ( B ) Expression levels of condensin I- and condensin II-specific subunits and their relationship to MYCN amplification or expression. The data were obtained from a published data set (GSE3960). The red line indicates low MYCN expression or no MYCN amplification, and the blue line indicates high MYCN expression or MYCN amplification.

Article Snippet: The following antibodies were used: anti-MYCN monoclonal antibodies (OP13, Calbiochem and NB200-109, Novus Biologicals); anti-MYCN monoclonal antibody (B8.4.B) (sc-53993, Santa Cruz); anti-SMC2 rabbit polyclonal antibodies (GTX10411, GeneTex; and ab10412, Abcam); anti-β-ACTIN monoclonal antibody (AC-15) (A5441, Sigma); anti-SMC4 rabbit polyclonal antibody (ab17958, Abcam); anti-CAP-D2 rabbit polyclonal antibody (A300-601A, Bethyl Laboratories); and anti-phospho-histone H2A.X (Ser139) mouse monoclonal antibody (JBW301, Millipore).

Techniques: Expressing, Amplification

Journal: Cell Cycle

Article Title: Inactivation of SMC2 shows a synergistic lethal response in MYCN -amplified neuroblastoma cells

doi: 10.4161/cc.27983

Figure Lengend Snippet: Figure 6. Schematic model showing the synergistic effects of SMC2 and MYCN in MYCN -amplified ( A ) and MYCN single copy ( B ) cells. This model is based on the proposed transcriptional regulation by MYCN and SMC2 ( C ).

Article Snippet: The following antibodies were used: anti-MYCN monoclonal antibodies (OP13, Calbiochem and NB200-109, Novus Biologicals); anti-MYCN monoclonal antibody (B8.4.B) (sc-53993, Santa Cruz); anti-SMC2 rabbit polyclonal antibodies (GTX10411, GeneTex; and ab10412, Abcam); anti-β-ACTIN monoclonal antibody (AC-15) (A5441, Sigma); anti-SMC4 rabbit polyclonal antibody (ab17958, Abcam); anti-CAP-D2 rabbit polyclonal antibody (A300-601A, Bethyl Laboratories); and anti-phospho-histone H2A.X (Ser139) mouse monoclonal antibody (JBW301, Millipore).

Techniques: Amplification