Activation marker staining. Mice were killed by CO 2 overdose at the times after infection indicated. Spleens were removed and disrupted by passage through a sieve and red blood cells were lysed by NH 4 Cl solution. 19 Two. (a) GSEA of microarray data for CD44 v + cell/ CD44 v − cell, ES cell/MEF (GSE16925 and GSE11274), and iPS cell/MEF (GSE16925) profiles. (b) Kaplan–Meier plots of overall survival according to ESRP1 (219121_s_at. The CD44 antigen is a cell-surface glycoprotein involved in cell–cell interactions, cell adhesion and migration. In humans, the CD44 antigen is encoded by the CD44 gene on Chromosome 11. CD44 has been referred to as HCAM. Alternative splicing of CD4. RNA by ESRP1 enhances lung colonization of metastatic cancer cell : Nature Communications : Nature Research. Cell culture. The murine breast cancer cell line 4. T1 (American Type Culture Collection, Rockville, MD) as well as CD4. CD4. 4v− cells sorted therefrom were cultured under 5% CO2 at 3. C in RPMI 1. 64. 0 medium (Sigma, St. Louis, MO) supplemented with 1. NR, 1. 68. FARN, and 4. T0. 7 cells (kindly provided by Link Genomics, Tokyo, Japan) were cultured under 5% CO2 at 3.
Polyclonal Antibody for studying CD44 in the Lymphocyte Signaling research area.C in Dulbecco's modified Eagle's medium- F1. Sigma) supplemented with 1. Mice. Wild- type Balb/c mice were obtained from CLEA Japan or Japan SLC (Tokyo, Japan). They were bred and maintained in the animal facility at Keio University according to institutional guidelines. All animal experiments were performed in accordance with protocols approved by the Ethics Committee of Keio University. Orthotopic transplantation and metastasis assay. Parental, CD4. 4v+, or CD4. CD44 is a cell surface HA-binding glycoprotein that is overexpressed to some extent by almost all tumors of epithelial origin and plays an important role in tumor initiation and metastasis. CD44 is a compelling marker for. Immunohistochemical analysis of human Non-Hodgkin's lymphoma using CD44 (156-3C11) Mouse mAb. Learn more about how we get our images. T1 cells (1×1. 05) were orthotopically injected into the fourth mammary gland of syngeneic 8- to- 1. Balb/c mice. At 3. Quantification of lung metastasis was performed by imaging as described below. Integri. Sense imaging. Twenty- four hours before imaging, mice were injected intravenously with 2 nmol of Integri. Sense 7. 50 (Vis. En Medical, Bedford, MA). Fluorescence imaging was performed with a Clairvivo. OPT in vivo fluorescence imager (Shimadzu), with excitation at 7. For quantitative comparisons, the total fluorescence signal intensity (pixel count) in a region of interest corresponding to each lung metastasis was measured. Tumour cell isolation and flow cytometry. T1. PT or 4. T1. LM cells were isolated from primary tumours or lung metastases, respectively, formed 3. T1 cells (1×1. 05) into the mammary gland of recipient mice. Primary tumours or lung metastases were digested for 4 to 5 h at 3. C in Ham's F1. 2 medium supplemented with 5% fetal bovine serum, penicillin (1. U ml−1), streptomycin (1. U ml−1), and hyaluronidase (1. U ml−1). Red blood cells were then lysed by the addition of NH4. Cl to a final concentration of 1. M at room temperature, and tissue fragments were dissociated by gentle pipetting first in the presence of 0. DNase I (0. 1 mg ml−1) for 2 min. All reagents were obtained from Stemcell Technologies (Vancouver, Canada). The mixture was then filtered through a 4. M 6- TG (Sigma) for 2 to 3 days and then subjected to FACS to isolate Lin− cells. Cultured cells were dissociated by exposure to enzyme- free, Hanks- based Cell Dissociation Buffer (GIBCO- Invitrogen, Tokyo, Japan). For flow cytometric analysis or FACS, single- cell suspensions were incubated with antibodies for 2. C. Antibodies included phycoerythrin- and Cy. Lin) CD3. 1, CD4. TER1. 19 (Bio. Legend, San Diego, CA, USA), phycoerythrin- conjugated antibodies to pan- CD4. IM7; e. Bioscience, San Diego, CA, USA), antibodies to x. CT (Abcam, Cambridge, UK), allophycocyanin- conjugated antibodies specific for mouse CD4. CD4. 4v. 9 (ref. 1. Apoptotic cells were excluded for all flow cytometric analysis and FACS by elimination of cells positive for staining with propidium iodide. Flow cytometric analysis and FACS were performed with a FACSCalibur instrument (BD Biosciences, Tokyo, Japan) and either a FACSAria Cell Sorter (BD Biosciences) or Mo. Flo Cell Sorter (Beckman Coulter, Tokyo, Japan), respectively. For measurement of ROS or GSH levels, cells were incubated with 1. M DCFH- DA or CFMDA (Invitrogen/Molecular Probes, Tokyo, Japan), respectively, for 1. C, washed twice with phosphate- buffered saline, and subjected to flow cytometric analysis. Immunoblot analysis. Immunoblot analysis was performed, as described previously. CD4. 4 (IM7; BD Pharmingen, Tokyo, Japan; 5. E- cadherin (BD Pharmingen; 1,0. ZO- 1, (Invitrogen, Tokyo, Japan; 1. Twist (H- 8. 1; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 2. N- cadherin (N- 1. Santa Cruz; 1,0. 00× dilution), Vimentin (BD Pharmingen; 2. Tubulin (Sigma; 1,0. ALDEFLUOR assay. An ALDEFLUOR kit (Stem. Cell Technologies) was used to detect cells with a high ALDH activity. Cells were suspended in ALDEFLUOR assay buffer containing the ALDH substrate BODIPY aminoacetaldehyde (BAAA) and incubated for 4. C. As a negative control, cells were incubated in the additional presence of diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor. Imaging mass spectrometry. T1 lung metastasis tissues snap- frozen in liquid nitrogen were dissected to prepare cryosections with 5- μm thickness by use of a cryostat (CM 1. Leica Microsystems, Wetzlar, Germany). The sections were thaw- mounted on indium–tin oxide slides (Bruker Daltonik Gmb. H, Bremen, Germany) and were dried in silica gel- containing plastic tubes and, then, sprayed with 9- aminoacridine (9- AA, 2. Me. OH) by use of a 0. Procon boy FWA Platinum; Mr Hobby, Tokyo, Japan) to conduct matrix- assisted laser desorption/ionization (MALDI) imaging mass spectrometry in negative ion mode. Adjacent sections were fixed with 4% buffered formalin (Nacalai Tesque, Kyoto, Japan) and stained with anti- CD4. All the MALDI imaging experiments was carried out in negative- ion mode using a prototype Mass Microscope (Shimadzu Corporation, Kyoto, Japan). The laser power was adjusted to the desired intensities. MALDI mass spectra were acquired under the conditions laser diameter 1. Regions of the tissue samples exposed to the laser irradiation were determined by light microscopic observation. GSEATo detect overlap in gene expression profiles among multiple sets of genes, we used GSEA with the Kolmogorov- Smirnov enrichment algorithm and 4. We first generated CD4. CD4. 4v− log. 2 ratio data from our microarray experiment and divided genes into those whose expression was upregulated (log. Second, we collected public microarray data (GSE1. GSE1. 69. 25) from the GEO database and calculated the P- value (t test) and log. ES cells/MEFs or i. PS cells/MEFs with the use of the SAM R- package. We extracted the genes with a > 2. SAM P- value of < 0. Third, up- or down- regulated gene sets in our CD4. CD4. 4v− microarray experiment were compared with the ES and i. PS cell signature gene sets by GSEA. Ch. IP sequencing and data analysis. Ch. IP was performed as previously described. CD4. 4v+ or CD4. 4v− 4. T1 cells were fixed with 1% formaldehyde, neutralized with 0. M glycine, and suspended in a lysis buffer containing 1. M Tris–HCl (p. H 8. M Na. Cl, 1% SDS, 1 m. M EDTA, and a protease inhibitor cocktail (Roche, Tokyo, Japan). The cell lysates were subjected to ultrasonic treatment (Sonifier 2. Branson, Kanagawa, Japan) and diluted with a solution containing 2. M Tris–HCl (p. H 8. M Na. Cl, 1 m. M EDTA, and 1% Triton X- 1. Protein A/G (Invitrogen)- bound antibodies to H3. K4me. 3 or to RNA polymerase II (both kindly provided by H. Kimura) or with those to H3. K2. 7me. 3 (Merck, Tokyo, Japan). Precipitated DNA was prepared according to the Illumina/Solexa Genomic DNA protocol (Illumina, San Diego, CA). The DNA was amplified by PCR (1. Genome Analyzer. DNA fragments of 3. M for loading on the flow cell. The DNA library (2 p. M) was applied to the flow cell with the use of the Cluster Station device (Illumina). Ch. IP sequencing (Ch. IP- seq) data were analysed, as described. In brief, images acquired from the Illumina/Solexa sequencer were processed through the bundled Solexa image extraction pipeline, which identified Polony positions, performed base- calling, and generated quality control (QC) statistics. Sequences were aligned with the human genome in the NCBI genomic database (UCSC mm. Sequences that mapped uniquely to the genome with two- base mismatches were selected. Sequences from all lanes for each Ch. IP sample were combined, extended 2. Genomic bins showing statistically significant Ch. IP- seq enrichment were identified by comparison with a Poissonian background model. For every 5. 00- bp (for H3. K4me. 3 and RNA polymerase II) or 1,0. H3. K2. 7me. 3) window, the mapped tag count for the Ch. IP sample (Cc) and that for the Ch. IP input (Ci) were used for calculation. Ec and Ei represent the estimated counts for 5. Ch. IP sample and Ch. IP input, respectively. The signal ratio was calculated as (Cc/Ec+1)/Max(1, Ci/Ei+1). These signals were visualized with Integrated Genome Browser software (Affymetrix, Santa Clara, CA, USA)5. Statistical analysis. Data are presented as means±s. Student's t test. A P- value of < 0.
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