Journal: STAR Protocols

Article Title: Engineering Elastic Nano- and Micro-Patterns and Textures for Directed Cell Motility

doi: 10.1016/j.xpro.2019.100013

Figure Lengend Snippet: Mechanical Rigidity of ICAM1 Nano-Topographies Controls T Cell On-Ridge Spreading and In-Groove Invasiveness Plasticity Balance (A) 3D super-resolution reconstruction of the ICAM1-functionalized PAA nano-topographic surface (G’=16 kPa). (B) Test super-resolution imaging (3D reconstruction) of human CD4+ T cells spreading and migrating along the ICAM1-coated nano-topographic surfaces on soft (16 kPa) and rigid (50 kPa) PAA surfaces. (C) Schematic of the T cell morphometric analysis and metrics: T cell height, projected area of spreading of entire cell interface ( S entire cell IF ) and projected area of in-groove invasive T cell interface ( S invasive IF ). (D and E) Mechanoregulation of T cell height, spreading area and invasiveness as indicated by T cell spreading assay on soft (G’=16 kPa) and rigid (G’=50 kPa) ICAM1 nano-textures. T cell spreading enhances on the rigid ICAM1, accompanied with T cell flattening, i.e. decrease of the T cell height. Results indicate a mechanically controlled dynamic balance between on-ridge T cell spreading and in-groove invasiveness, as shown on the schematic panel (E). I.e. T cell in-groove invasiveness structurally competes with on-ridge spreading, indicating that on-ridge spreading is mechanically enhanced and out-balances in-groove invasiveness on the rigid (G’=50 kPa) ICAM1 nano-topography. Alternatively, soft (G’=16 kPa) ICAM-1 nano-textures are unable to promote the mechanically sensitive on-ridge T cell spreading, shifting the balance towards steric in-groove T cell invasiveness. Data on the plots on (D) are as follows: boxes - means, Q1 and Q3; whiskers - max and min, X - medians; p values - one way ANOVA test. Experimental data collected in triplicates, total n>50.

Article Snippet: ICAM1, Human Protein, Recombinant, hIgG1-Fc.His Tag, Active , Sino Biological, China , Cat#10346-H03H-50.

Techniques: Imaging

Journal: STAR Protocols

Article Title: Engineering Elastic Nano- and Micro-Patterns and Textures for Directed Cell Motility

doi: 10.1016/j.xpro.2019.100013

Figure Lengend Snippet:

Article Snippet: ICAM1, Human Protein, Recombinant, hIgG1-Fc.His Tag, Active , Sino Biological, China , Cat#10346-H03H-50.

Techniques: Recombinant, Electrophoresis, Concentration Assay, Labeling, Saline, Modification, Cell Isolation, Derivative Assay, Software

Journal: Journal of Virology

Article Title: Human T-Lymphotropic Virus Type 1 Transcription and Chromatin-Remodeling Complexes

doi: 10.1128/jvi.00851-09

Figure Lengend Snippet: FIG. 1. Association of Tax with CBP/p300 and SWI/SNF complexes. (A) (Left) Schematic representation of chromatographic steps using C8166 nuclear extracts. Nuclear extracts were passed through P11, DEAE Sephacel, Mono S, and Superose 6 sizing columns to obtain Tax and associated factors. (Right) Chromatogram of the fast protein liquid chromatography (FPLC) fractions, where peak A shows Superose 6 at 0.7 M salt and peak B shows 0.15 M salt. Fractions 13 to 17, which contain a high-molecular-weight complex, are fairly stable at either salt concentration. (B) Western blot analysis of Superose 6 column fractions using antibodies against Pol II, CBP/p300, Tax, and BRG1. Fractions were loaded and isolated at 0.7 M salt. Tax eluted at three distinct locations, including a small complex (120 kDa), a medium complex (1 MDa), and a large complex ( 2.2 MDa). (C) Immunoprecipitation using anti-Tax or control antibodies (IgG), followed by Western blot analysis for antibodies against BRG1 and CBP/p300. p300 is slightly larger than CBP (molecular weight, 265,000 [265K]) (68). A total of 330 g of each fraction (fractions 15 and 21) was used for immunoprecipitation with 1.0 g of the primary antibody. (D) Reciprocal IP of fraction 15 with anti-BRG1, as well as IP with anti-CBP/p300 from fraction 21, followed by Western blotting for Tax.

Article Snippet: Antibodies against p300 (sc584), CBP (sc1211), and BRG1 (sc10768) were obtained from Santa Cruz.

Techniques: Fast Protein Liquid Chromatography, High Molecular Weight, Concentration Assay, Western Blot, Isolation, Immunoprecipitation, Control, Molecular Weight

Journal: Journal of Virology

Article Title: Human T-Lymphotropic Virus Type 1 Transcription and Chromatin-Remodeling Complexes

doi: 10.1128/jvi.00851-09

Figure Lengend Snippet: FIG. 4. Determination of translational positioning of HTLV-1 nucleosomes and absence of various histones in transfected H1GFP cells and PBMCs. (A) DNase I treatment of permeabilized H1GFP (HTLV-1 LTR-GFP) cells for mapping of DNase I-hypersensitive sites. Cells were grown to 70% confluence and were then either left untransfected (lane 1); transfected with increasing amounts of Tax alone (lane 2, 5 g; lane 3, 10 g), five wild-type BRG1-specific siRNAs (20 g) plus Tax (10 g) (lane 4), or five mutant BRG1 siRNAs (20 g) plus Tax (10 g) (lane 5); or treated with NaB (10 M). Forty-eight hours later, cells were prepared for a DNase I hypersensitivity assay, size fractionated on an agarose gel, and transferred to a membrane. The nick-translated H1GFP fragment (5 g) was used as a hybridization probe for Southern blotting. (B) Effect of Tax on histone eviction after Tax treatment. H1GFP cells were either left untreated or transfected with a Tax plasmid using Lipofectamine. Forty-eight hours later, samples were processed for ChIP using various antibodies against histones. Primers for the 1 start site (Nuc 1 area) spanned the area from 64 to 82. (C) Human PBMCs from two healthy donors were first purified by Ficoll-Paque centrifugation and then activated with PHA (10 g/ml) and recombinant human IL-2 (50 U/ml). The cells were transfected with double-CsCl-purified ACH.WT (40 g) using the Amaxa reagent. The cells were then cultured in cRPMI supplemented with PHA (5 g/ml) and IL-2 for 6 weeks, after which they were cultured in the same medium without PHA. At various time points, cell culture supernatants were collected for p19 determination (data not shown), and relative cellular viability was assayed by MTT conversion assays on 100-l aliquots of cells. Cells that survived for 70 days were 90% infected with HTLV-1. O.D. 590, optical density at 590 nm. (D) ChIP assays with samples from donor 1 PBMCs transfected with the wild-type clone ACH.WT. ChIPs were performed with 70-day samples using various antibodies. PCR amplification was performed for both the Nuc 1 (1 area) and Nuc 8 (Gag) regions.

Article Snippet: Antibodies against p300 (sc584), CBP (sc1211), and BRG1 (sc10768) were obtained from Santa Cruz.

Techniques: Transfection, Mutagenesis, Agarose Gel Electrophoresis, Membrane, Hybridization, Southern Blot, Plasmid Preparation, Centrifugation, Recombinant, Cell Culture, Infection

Journal: Journal of Virology

Article Title: Human T-Lymphotropic Virus Type 1 Transcription and Chromatin-Remodeling Complexes

doi: 10.1128/jvi.00851-09

Figure Lengend Snippet: FIG. 5. Effect of BRG1 on HTLV-1 LTR Tax-activated transcription in mutant cells. (A and B) Titration of Tax (0.1, 1, 3, and 5 g) either alone (lanes 2 to 5) or with a BRG1 plasmid (5 g) (lanes 6 to 9). A total of 5 106 C33A (A) or SW13 (B) cells were transfected for 3 days and were subsequently processed for CAT assays (50 g of total extract). Lanes 1 serve as a negative control (HTLV-1-LTR-CAT and no Tax), and lanes 10 also serve as a control for BRG1 (5 g of a BRG1 dominant-negative mutant [38]). BRG1 enhanced the level of activation dramatically even with minimal concentrations of Tax (i.e., lanes 6 in both panels). (C) LTR-CAT was transfected either with BRG1 alone or with BRG1 plus Tax (3 g) into either C33A or SW13 cells, as for panels A and B. The data represent three independent replicates. %Conversion, %conversion from unacetylated to mono- or diacetylated chloramphenicol.

Article Snippet: Antibodies against p300 (sc584), CBP (sc1211), and BRG1 (sc10768) were obtained from Santa Cruz.

Techniques: Mutagenesis, Titration, Plasmid Preparation, Transfection, Negative Control, Control, Dominant Negative Mutation, Activation Assay

Journal: Journal of Virology

Article Title: Human T-Lymphotropic Virus Type 1 Transcription and Chromatin-Remodeling Complexes

doi: 10.1128/jvi.00851-09

Figure Lengend Snippet: FIG. 6. Lack of HTLV-1 viral replication in BRG1 mutant cells. (A) 293T cells, used as a control, were transfected with the HTLV-1 wild-type clone, ACH.WT. Supernatants from cells infected with HTLV-1 MT2 (20 l) and treated with TNF or from 293T cells transfected with an empty vector (supernatants after 4 days) served as positive and negative controls, respectively. Optimal HTLV-1 replication occurred on day 3. (B and C) Both SW13 and C33A BRG1 mutant cells were transfected with either the pcDNA3 vector (data not shown) or the ACH.WT plasmid (20 g), and experiments were carried out to day 12. Day 1 is a negative control with no transfection. Viral RT was present only in the supernatants of BRG1-transfected cells and peaked at day 12 in either cell type. All experiments in panels B and C were performed in triplicate. Western blotting for BRG1 and actin of samples from SW13 or C33A cells transfected with a mock plasmid (left lanes 1, 3, 9, and 12) or a BRG1 plasmid on days 3, 6, 9, and 12 (right lanes and insets). (D) C33A cells from panel C at day 12 were processed for ChIP experiments using either an IgG control or the anti-Pol II antibody 8WG16. The LTR primers spanned the region from 350 to 250 (U3/R/U5), and the Env primers spanned the region from 8990 to 9450.

Article Snippet: Antibodies against p300 (sc584), CBP (sc1211), and BRG1 (sc10768) were obtained from Santa Cruz.

Techniques: Mutagenesis, Control, Transfection, Infection, Plasmid Preparation, Negative Control, Western Blot

Journal: Journal of Virology

Article Title: Human T-Lymphotropic Virus Type 1 Transcription and Chromatin-Remodeling Complexes

doi: 10.1128/jvi.00851-09

Figure Lengend Snippet: FIG. 7. Effects of various siRNAs against SWI/SNF components on the wild-type HTLV-1 LTR. (A) BRG1-associated complexes. Brahma- related gene-1 (BRG1) is a catalytic subunit found in a variety of chromatin-remodeling complexes, including the SWI/SNF complexes BAF and PBAF, as well as WINAC, NCoR, mSin3A/HDAC, and NUMAC. The various complexes share other subunits that have a variety of activities, color coded as follows: green, core components; pink, nuclear receptor association; gold, DNA replication; light green, actin-related complexes; blue, Ini1/Baf47/SNF5; purple, other functions (63). As chromatin remodelers, these complexes can act either as transcriptional activators, as transcriptional repressors, or as both. For example, NUMAC acts as a transcriptional activator, while the mSin3A/HDAC complex acts as a transcriptional repressor. SWI/SNF complexes can act as either repressors or activators. (B) RT assay of supernatants (10 l) from 293T cells (positive control), BRG1 mutant (TSUPR1, C33A, and SW13) cells, and Baf180 mutant (HCC1143) cells. Cells were transfected with the ACH.WT plasmid (20 g) and were grown for 6 days. Supernatants were collected at day 6 and were then processed for the RT assay. BRG1 (10 g), along with ACH.WT, was also transfected into five cell types (lanes 2, 4, 6, 8, and 10). (C) 293T cells were transfected with various siRNAs, including siBRG1, siBRM, siBaf250, and siBaf180 (300 nM), along with ACH.WT (20 g). Samples were processed for RT at day 6. (Inset) Cell pellets were processed for Western blotting with antibodies against BRG1 and various other components. The actin panel represents cells transfected with siBRG1. Data represent one replicate of three independent experiments.

Article Snippet: Antibodies against p300 (sc584), CBP (sc1211), and BRG1 (sc10768) were obtained from Santa Cruz.

Techniques: Positive Control, Mutagenesis, Transfection, Plasmid Preparation, Western Blot