Journal: Scientific Reports

Article Title: Comparative analysis of linker histone H1, MeCP2, and HMGD1 on nucleosome stability and target site accessibility

doi: 10.1038/srep33186

Figure Lengend Snippet: Nucleosomal construct used in FRET study ( a ) DNA construct used in our FRET studies ; contains a 601 NPS, the Cy3 fluorophore (position 43), a Gal4 binding site shown as a blue bar (position 48–76), and an extra 50 bp linker DNA at the entry/exit ends of the 601 NPS. ( b ) Structure of Cy3-Cy5 labeled nucleosome construct adapted from , showing the labels in relation to the DNA and histone octamer.

Article Snippet: After salt dialysis the reconstituted mononucleosomes were purified on a 5–30% sucrose gradient centrifuged at 41,000 × rpm for 22 h. For 17-mer chromatin/chromatosome array: human histone octamer (a kind gift from the Luger laboratory) and specified CAP was added to DdeI (NEB) digested 17-mer plasmid at a stoichiometry of 23(CAP):23(histone octamer):1(17-mer array).

Techniques: Construct, Binding Assay, Labeling

Journal: bioRxiv

Article Title: Intrinsic elasticity of nucleosomes is encoded by histone variants and calibrated by their binding partners

doi: 10.1101/392787

Figure Lengend Snippet: Alignment of various plant, fungal, and animal species shows that within the poorly conserved central domain and the well conserved CENP-C motif the RR(S/T)nR motif and the WW/YW motif are highly conserved. These two motifs are separated by 7 residues, creating a conserved H2A/H2B acid patch and C-terminal tail of CENP-A binding motif (RR(S/T)nRnRnnP(L/S)nn(WW/YW).

Article Snippet: In vitro reconstitution of CENP-A nucleosome arrays (CENP-A/H4 cat#16-010 and H2A/H2B cat#15-0311, EpiCypher, Research Triangle Park, NC) and H3 (H3/H4 cat#16-0008 and H2A/H2B cat#15-0311, EpiCypher Research Triangle Park, NC) on a 3kb plasmid containing a single 601 sequence (pGEM3Z-601 from Addgene #26656) were performed as previously described ( , ).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Intrinsic elasticity of nucleosomes is encoded by histone variants and calibrated by their binding partners

doi: 10.1101/392787

Figure Lengend Snippet: ( A ) Roundness was measured of either H3 or CENP-A mononucleosomes. A value of 1 would indicate that a nucleosome particle lays flat on the mica surface, whereas a value of 0.5 would indicate a nucleosome particle laying on its side. Almost all nucleosomal particles lay flat. ( B ) Young’s modulus was measured across H3 or CENP-A mononucleosomes to assess whether a nucleosome particle is uniformly elastic. No significant difference in Young’s moduli was observed across either nucleosome.

Article Snippet: In vitro reconstitution of CENP-A nucleosome arrays (CENP-A/H4 cat#16-010 and H2A/H2B cat#15-0311, EpiCypher, Research Triangle Park, NC) and H3 (H3/H4 cat#16-0008 and H2A/H2B cat#15-0311, EpiCypher Research Triangle Park, NC) on a 3kb plasmid containing a single 601 sequence (pGEM3Z-601 from Addgene #26656) were performed as previously described ( , ).

Techniques:

Journal: bioRxiv

Article Title: Intrinsic elasticity of nucleosomes is encoded by histone variants and calibrated by their binding partners

doi: 10.1101/392787

Figure Lengend Snippet: ( A ) Histone protein concentration was determined by Coomassie staining. ( B ) BioAnalyzer results from 10, 30, and 90 seconds of MNase digested reconstituted H3 and CENP-A nucleosomes on 3 kbp plasmids a classic chromatin ladder. ( C ) Representative in air AFM images of H3 and CENP-A reconstituted chromatin.

Article Snippet: In vitro reconstitution of CENP-A nucleosome arrays (CENP-A/H4 cat#16-010 and H2A/H2B cat#15-0311, EpiCypher, Research Triangle Park, NC) and H3 (H3/H4 cat#16-0008 and H2A/H2B cat#15-0311, EpiCypher Research Triangle Park, NC) on a 3kb plasmid containing a single 601 sequence (pGEM3Z-601 from Addgene #26656) were performed as previously described ( , ).

Techniques: Protein Concentration, Staining

Journal: bioRxiv

Article Title: Intrinsic elasticity of nucleosomes is encoded by histone variants and calibrated by their binding partners

doi: 10.1101/392787

Figure Lengend Snippet: ( A ) To obtain Young’s modulus values from simulation, we measured the in silico dimensions of nucleosomes by compression of an encapsulating cylinder programmed to stop at stiffer surfaces resistant to collapse. From the heights, h = z max – z min , and the radii, r max , of the resulting minimal cylinders we then calculated the average and change in height (h avg, Δh), and radius (r avg, Δr) of each system. ( B ) We treated the nucleosomes as elastic homogenous cylinders, calculated the energy of deformation, and retrieved the Young’s modulus of a cylinder vibrating at equilibrium in a thermal bath.

Article Snippet: In vitro reconstitution of CENP-A nucleosome arrays (CENP-A/H4 cat#16-010 and H2A/H2B cat#15-0311, EpiCypher, Research Triangle Park, NC) and H3 (H3/H4 cat#16-0008 and H2A/H2B cat#15-0311, EpiCypher Research Triangle Park, NC) on a 3kb plasmid containing a single 601 sequence (pGEM3Z-601 from Addgene #26656) were performed as previously described ( , ).

Techniques: In Silico

Journal: bioRxiv

Article Title: Intrinsic elasticity of nucleosomes is encoded by histone variants and calibrated by their binding partners

doi: 10.1101/392787

Figure Lengend Snippet: ( A ) Height and diameter predictions from the computational modeling experiment described in . ( B ) CENP-C CD modestly increases height of in vitro reconstituted CENP-A nucleosomes H3 and CENP-A nucleosome were in vitro reconstituted, and by in fluid AFM, we measured their dimensions (height, diameter, and volume). The height distribution is shown in the violin plot containing a bar plot. CENP-A nucleosomes are ever so slightly smaller than H3 nucleosomes. The addition of CENP-C CD fragment, which can only bind CENP-A nucleosomes, we observed an increase in height and in a dose-dependent manner its volume.

Article Snippet: In vitro reconstitution of CENP-A nucleosome arrays (CENP-A/H4 cat#16-010 and H2A/H2B cat#15-0311, EpiCypher, Research Triangle Park, NC) and H3 (H3/H4 cat#16-0008 and H2A/H2B cat#15-0311, EpiCypher Research Triangle Park, NC) on a 3kb plasmid containing a single 601 sequence (pGEM3Z-601 from Addgene #26656) were performed as previously described ( , ).

Techniques: In Vitro

Journal: bioRxiv

Article Title: Intrinsic elasticity of nucleosomes is encoded by histone variants and calibrated by their binding partners

doi: 10.1101/392787

Figure Lengend Snippet: Four representative force curves for H3 nucleosomes, CENP-A nucleosomes, CENP-A nucleosomes with 2-fold excess CENP-C CD fragments, and CENP-A nucleosomes with 4-fold excess CENP-C CD fragments are shown.

Article Snippet: In vitro reconstitution of CENP-A nucleosome arrays (CENP-A/H4 cat#16-010 and H2A/H2B cat#15-0311, EpiCypher, Research Triangle Park, NC) and H3 (H3/H4 cat#16-0008 and H2A/H2B cat#15-0311, EpiCypher Research Triangle Park, NC) on a 3kb plasmid containing a single 601 sequence (pGEM3Z-601 from Addgene #26656) were performed as previously described ( , ).

Techniques:

Journal: bioRxiv

Article Title: Intrinsic elasticity of nucleosomes is encoded by histone variants and calibrated by their binding partners

doi: 10.1101/392787

Figure Lengend Snippet: Bar plot summarizing the Young’s modulus values showing that CENP-A nucleosomes become stiffer upon addition of 2-fold excess CENP-C CD (two-sided t-test p =0.015), and even stiffer upon addition of 4-fold excess CENP-C CD binding (two-sided t-test p <0.001). ∼1000 force curves were measured per condition.

Article Snippet: In vitro reconstitution of CENP-A nucleosome arrays (CENP-A/H4 cat#16-010 and H2A/H2B cat#15-0311, EpiCypher, Research Triangle Park, NC) and H3 (H3/H4 cat#16-0008 and H2A/H2B cat#15-0311, EpiCypher Research Triangle Park, NC) on a 3kb plasmid containing a single 601 sequence (pGEM3Z-601 from Addgene #26656) were performed as previously described ( , ).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Intrinsic elasticity of nucleosomes is encoded by histone variants and calibrated by their binding partners

doi: 10.1101/392787

Figure Lengend Snippet: ( A ) Qualitative assessment of in vitro reconstituted chromatin showed that only CENP-A chromatin clustered in the presence of CENP-C CD fragment. ( B ) To determine if the CENP-C CD fragment used in the in vitro experiments could induce CENP-C chromatin compaction, we added CENP-C CD for 30 minutes to isolated free CENP-A chromatin from HeLa cells. Compacted chromatin was scored over the total number of nucleosome arrays. ( C ) Similar analysis was performed on unbound CENP-A chromatin extracted from cells that either did (CENP-C OE ) or did not (WT) overexpress CENP-C. ( D ) Western blot of serial N-ChIP probing for RNAP2 and various centromere and chromatin markers. Quantification of ( E ) RNAP2 levels were determined by LiCor’s software. The bar graphs represent three independent experiments. ( F ) Working model of CENP-C (yellow) overexpression inducing CENP-A chromatin (red) cross-array clustering thereby reducing access to RNAP2 (blue).

Article Snippet: In vitro reconstitution of CENP-A nucleosome arrays (CENP-A/H4 cat#16-010 and H2A/H2B cat#15-0311, EpiCypher, Research Triangle Park, NC) and H3 (H3/H4 cat#16-0008 and H2A/H2B cat#15-0311, EpiCypher Research Triangle Park, NC) on a 3kb plasmid containing a single 601 sequence (pGEM3Z-601 from Addgene #26656) were performed as previously described ( , ).

Techniques: In Vitro, Isolation, Western Blot, Software, Over Expression

Journal: bioRxiv

Article Title: Intrinsic elasticity of nucleosomes is encoded by histone variants and calibrated by their binding partners

doi: 10.1101/392787

Figure Lengend Snippet: ( A ) To determine the Young’s modulus of CENP-A and H3 nucleosome arrays, we in vitro reconstituted H3 and CENP-A nucleosome arrays by salt dialysis, followed by nanoindentation force spectroscopy. ( B ) Bar plot summarizing the Young’s modulus values showing that CENP-A nucleosomes are more elastic than H3 nucleosomes but become stiffer upon addition of CENP-C CD (two-sided t-test p <0.0001). ∼1000 force curves were measured per condition.

Article Snippet: In vitro reconstitution of CENP-A nucleosome arrays (CENP-A/H4 cat#16-010 and H2A/H2B cat#15-0311, EpiCypher, Research Triangle Park, NC) and H3 (H3/H4 cat#16-0008 and H2A/H2B cat#15-0311, EpiCypher Research Triangle Park, NC) on a 3kb plasmid containing a single 601 sequence (pGEM3Z-601 from Addgene #26656) were performed as previously described ( , ).

Techniques: In Vitro, Spectroscopy

Journal: bioRxiv

Article Title: Intrinsic elasticity of nucleosomes is encoded by histone variants and calibrated by their binding partners

doi: 10.1101/392787

Figure Lengend Snippet: ( A ) The distance between the center of mass (COM) of histone dimers is shown in red for CENP-A, blue for CENP-A + 1 CENP-C CD , and in orang for CENP-A + 2 CENP-C CD . Two CENP-C CD fragment exaggerated the COM distances compared to a single CENP-C CD fragment, which means that 2 CENP-C CD further induces a global loss of CENP-A nucleosome flexibility. ( B ) The free energy landscape of CENP-A nucleosomes alone or CENP-A nucleosome with CENP-C CD fragment was determined by principle component analysis. CENP-A nucleosomes display a rugged free energy landscape, which is locked down when CENP-C CD is bound, increasing the connectivity of the energetic minima. All-atom computational modeling of DNA gyre gapping or DNA gyre sliding of CENP-A nucleosome alone or bound to either 1 or 2 CENP-C CD fragments. ( C ) Residue root mean square fluctuations (RMSF) shows freezing of local flexibility in the CENP-A nucleosome shown in red, 1 CENP-C CD bound shown in blue, and 2 CENP-C CD bound shown in orange. In the region of CENP-C CD binding, the first heterotypic half on the top panel, CENP-C is seen to freeze the acidic patch and the loop 1 region of CENP-A. One CENP-C CD creates asymmetry, especially at the C-terminal end of H2A and H2B, this is abrogated when the second CENP-C CD is bound. Dashed lines separate individual histones.

Article Snippet: In vitro reconstitution of CENP-A nucleosome arrays (CENP-A/H4 cat#16-010 and H2A/H2B cat#15-0311, EpiCypher, Research Triangle Park, NC) and H3 (H3/H4 cat#16-0008 and H2A/H2B cat#15-0311, EpiCypher Research Triangle Park, NC) on a 3kb plasmid containing a single 601 sequence (pGEM3Z-601 from Addgene #26656) were performed as previously described ( , ).

Techniques: Binding Assay

Journal: Oncogene

Article Title: Int6 regulates both proteasomal degradation and translation initiation and is critical for proper formation of acini by human mammary epithelium.

doi: 10.1038/onc.2010.445

Figure Lengend Snippet: Figure 5 Int6-dependent SRC3 degradation in MCF10A cells. (a) INT6 was silenced as in Figure 1a, and cell lysates were collected at two time points after transfection and analyzed by reverse-phase protein array. (b) After transfection by INT6 siRNA#1 for 24 h, SRC3 levels in mitotic (M) and interphase (I) cells were examined by western blots. Actin was the loading control. Int6 levels were reduced by approximately 80% in MCF10A cells with a concurrent four-fold increase of SRC3 in mitotic cells. Note that the presence of mitotic cells was confirmed by western blot using an anti-phospho-histone H3 antibody. (c) INT6 expression was repressed in MCF10A cells as in (b), and these cells were blocked in mitosis by thymidine/nocodazole. Cycloheximide (200 mg/ml) was added to inhibit denovo protein synthesis, and time points were analyzed by western blot. These time points are shown in a semi-log plot, in which Y-axis shows the decline of protein levels relative to those at t ¼ 0, which is set to 1. Error bars were calculated from three separate experiments. (d) Int6 was immunoprecipitated from MCF10A cells and pre-immune serum was used as the antibody control. Co-precipitated proteins were analyzed by western blots. Actin was examined as a specificity control. (e) Cells lysates from MCF7, HeLa, and MCF10A cells were examined by western blot to detect indicated proteins. Actin was the loading control.

Article Snippet: Antibodies against phospho-histone H3 (1:1,000), total histone H3 (1:1,000), cleaved caspase 3 (1:1,000) and actin (1:10,000) were from Cell Signaling (Boston, MA, USA).

Techniques: Transfection, Protein Array, Western Blot, Control, Expressing, Immunoprecipitation

Journal: Cancer cell

Article Title: MAST1 drives cisplatin resistance in human cancers by rewiring cRaf independent MEK activation

doi: 10.1016/j.ccell.2018.06.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-phospho-Histone gamma H2AX (S139) (clone 20E3) antibody , Cell Signaling Technology , Cat#9718; RRID: AB_2118009.

Techniques: Recombinant, Negative Control, Viability Assay, Kinase Assay, Extraction, Ab Array, Activation Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription, shRNA, Sequencing, Plasmid Preparation, Software

Journal: Cancers

Article Title: DUOX2, a New Biomarker for Disseminated Gastric Cancer’s Response to Low Dose Radiation in Mice

doi: 10.3390/cancers13164186

Figure Lengend Snippet: TCGA data of DUOX2 mRNA expression in human Stomach adenocarcinoma (STAD) tumors. ( A ) Levels of DUOX2 mRNA expression in 34 Normal and 415 Primary stomach adenocarcinoma. ( B ) Levels of DUOX2 mRNA expression based on tumor grade. The number of samples of each tumor grade are indicated in parenthesis. ( C ) Levels of DUOX2 mRNA expression based on nodal metastasis status. ( D ) Expression of DUOX2 mRNA based on individual cancer stages. Data provided by the UALCAN web resource for analyzing cancer OMICS data ( http://ualcan.path.uab.edu/index.html , accessed on 28 July 2021). Significance of difference estimated by Student’s t -test considering unequal variance. * p < 0.05.

Article Snippet: Human stomach adenocarcinoma tissue arrays (37 samples) with progressive disease were obtained from Pantomics, Inc. (Cat no, STC 962, Richmond, CA, USA) and analyzed by immunohistochemistry for DUOX2 (mouse monoclonal MIL-MABN787, 1/300, Millipore Sigma, St-Louis, MO, USA), and macrophages (CD68, mouse monoclonal EBS-14-0688-82, 1/1000).

Techniques: Expressing

Journal: Cancers

Article Title: DUOX2, a New Biomarker for Disseminated Gastric Cancer’s Response to Low Dose Radiation in Mice

doi: 10.3390/cancers13164186

Figure Lengend Snippet: Immuno-Histo-Chemistry (IHC) in 37 human adenocarcinoma samples. ( A ) Staining scored blinded by a pathologist at Pantomics, Inc. Staining > 2.8 was considered strong DUOX2 positive, score of “0” was considered DUOX2 negative. Percentage of strong staining and negative DUOX2 staining are indicated. ( B ) Example of heterogeneous (positive and negative) DUOX2 staining in well differentiated/low grade gastric adenocarcinoma (Malignant 1). Red arrows indicate cells expressing DUOX2. ( C ) Expanded view of inset from ( B ). ( D ) Example of strong DUOX2 staining in poorly differentiated/high grade stomach adenocarcinoma. ( E ) Intensity of CD68 staining in DUOX2 negative and positive samples. Student’s t -test, *** p = 0.0005. ( F ) CD68, red arrows indicate macrophages tumor and stroma infiltration in same tissue as ( B ) expressing DUOX2. ( G ) Expanded view of inset from ( F ). ( H ) IHC in poorly differentiated/high grade stomach adenocarcinoma showing strong CD68 staining. Images dimensions are 450 × 383 pixels (W × H).

Article Snippet: Human stomach adenocarcinoma tissue arrays (37 samples) with progressive disease were obtained from Pantomics, Inc. (Cat no, STC 962, Richmond, CA, USA) and analyzed by immunohistochemistry for DUOX2 (mouse monoclonal MIL-MABN787, 1/300, Millipore Sigma, St-Louis, MO, USA), and macrophages (CD68, mouse monoclonal EBS-14-0688-82, 1/1000).

Techniques: Staining, Expressing

Journal: Cancers

Article Title: DUOX2, a New Biomarker for Disseminated Gastric Cancer’s Response to Low Dose Radiation in Mice

doi: 10.3390/cancers13164186

Figure Lengend Snippet: Stat-1 response to mDCF and LDFRT in NCI-N87 cells. ( A ) Western blot analysis of total Stat-1, and Stat-1 phosphorylated at Serine 727 (p-Ser727) and Tyrosine 701 (p-Tyr701). Cells were exposed to the indicated treatment and pre-treated (+) or not (−) with the Src kinase inhibitor Dasatinib. ( B – D ) Fold induction measured by densitometry and normalized to Actin and the untreated sample (Vehicle). ( E ) Expression of Stat1 in normal and stomach adenocarcinoma (STAD) from TCGA samples accessed on 28 July 2021. . Student’s t -test, *** p < 0.0005.

Article Snippet: Human stomach adenocarcinoma tissue arrays (37 samples) with progressive disease were obtained from Pantomics, Inc. (Cat no, STC 962, Richmond, CA, USA) and analyzed by immunohistochemistry for DUOX2 (mouse monoclonal MIL-MABN787, 1/300, Millipore Sigma, St-Louis, MO, USA), and macrophages (CD68, mouse monoclonal EBS-14-0688-82, 1/1000).

Techniques: Western Blot, Expressing

Journal: Cancer cell

Article Title: MAST1 drives cisplatin resistance in human cancers by rewiring cRaf independent MEK activation

doi: 10.1016/j.ccell.2018.06.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-Histone H3 (clone 96C10) antibody , Cell Signaling Technology , Cat#3638; RRID: AB_1642229.

Techniques: Recombinant, Negative Control, Viability Assay, Kinase Assay, Extraction, Ab Array, Activation Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription, shRNA, Sequencing, Plasmid Preparation, Software