cell lines k562 cells american type culture collection n a kbm5 cells jin  (ATCC)

Journal: Cell reports

Article Title: Highly Parallel Quantification and Compartment Localization of Transcription Factors and Nuclear Proteins.

doi: 10.1016/j.celrep.2020.01.096

Figure Lengend Snippet: Figure 3. Immunofluoresence Reveals Extraction Kinetics and Validates CHESS-DIA Measurements (A) K562 nuclei were treated to extract each fraction and then fixed after a 5-min incubation. Fixed nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI) and antibodies recognizing LaminB1 and GATA1 and were imaged, and the boxplot represents a quantification of 100 cells. DIA data are shown at the peptide level for all GATA1 peptides detected in the library. Boxes represent the 25th, 50th, and 75th percentiles, and whiskers represent the minimum and maximum values. (B) Immunofluoresence-based quantification for 100 cells per time point. Markings represent the mean and standard error. (C) Same as (A), except for IF and DIA data for XPO1. Boxes represent the 25th, 50th, and 75th percentiles, and whiskers represent the minimum and maximum values. (D) Same as (A), except for IF and DIA data for BRG1. Student’s t tests were used for statistical tests; ***p < 0.001. Boxes represent the 25th, 50th, and 75th percentiles, and whiskers represent the minimum and maximum values.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies HDAC1 antibody, rabbit polyclonal Active Motif Cat#40967; RRID:AB_2614948 HDAC2 antibody, mouse monoclonal Active Motif Cat#39533; RRID:AB_2614959 BRD2 antibody, rabbit polyclonal Active Motif Cat#61797; RRID:AB_2793770 BRD3 antibody, rabbit polyclonal Active Motif Cat#61489; RRID:AB_2737028 BRD4 antibody, rabbit monoclonal Bethyl Cat#A700-004; RRID:AB_2631885 GATA1 antibody, rabbit monoclonal Cell Signaling Technology Cat#3535; RRID:AB_2108288 BRG1 antibody, mouse monoclonal Santa Cruz Biotechnology Cat#sc-17796; RRID:AB_626762 CRM1 (XOP1) antibody, mouse monoclonal Santa Cruz Biotechnology Cat#sc-74454; RRID:AB_1122704 Histone H3K27ac antibody, mouse monoclonal Active Motif Cat#39085; RRID:AB_2793305 Histone H3K27ac antibody, rabbit polyclonal Active Motif Cat#39133; RRID:AB_2722569 Lamin B Antibody, goat polyclonal Santa Cruz Biotechnology Cat#sc-6216; RRID:AB_648156 CTCF antibody, rabbit polyclonal Cell Signaling Technology Cat#2899; RRID:AB_2086794 CREBBP antibody, rabbit polyclonal Sigma Aldrich Cat#HPA055861; RRID:AB_2682948 HP1a antibody, rabbit polyclonal Cell Signaling Technology Cat##2616; RRID:AB_2070987 Chemicals, Peptides, and Recombinant Proteins dBET1 Tocris Cat#6327 PPS silent surfactant Expedeon Cat#21011 Pierce Trypsin Protease, MS Grade Thermo Fisher Cat#90057 Pierce Peptide Retention Time Calibration Mixture Thermo Fisher Cat#88320 Deposited Data Transcription Factor Annotations Lambert et al., 2018 https://doi.org/10.1016/j.cell.2018.01.029 MOLM13 nuclear proteomics data Winter et al., 2015 https://doi.org/10.1126/science.aab1433 ENCODE K562 data ENCODE Encyclopedia, Version 4 DNaseI: ENCSR000EOY RNaseq: ENCSR109IQO CTCF ChIP-seq: ENCFF389ELU Nuclear fraction proteomics data Torrente et al., 2011 N/A Gene Ontology Panther database version 14.0 Protein-protein interaction data BioGRID database version 3.5.169 Subcellular immunofluoresence data The Human Protein Atlas version 18.0 Experimental Models: Cell Lines K562 American Type Culture Collection CCL-243

Techniques: Extraction, Incubation, Staining

Journal: Blood Cancer Journal

Article Title: Improved anti-leukemia activities of adoptively transferred T cells expressing bispecific T-cell engager in mice

doi: 10.1038/bcj.2016.38

Figure Lengend Snippet: Blinatumomab BiTEs were secreted from RNA-transferred T cells and bound to T cells for tumor recognition. The T cells were electroporated with an RNA encoding CAR19 (CAR RNA), blinatumomab BiTEs (Blina-RNA) or GFP (at an RNA dose of 10 μg of RNA per 0.1 ml of T cells per electroporation). Eighteen hours after electroporation, the T cells were stained with a goat anti-mouse IgG Fab (mIgG Fab) to detect the expression of the CAR or blinatumomab on the T-cell surface (gated on CD3 + T cells) ( a ). Eighteen hours after electroporation, the CAR RNA or BiTE RNA T cells alone or mixed with an equal amount of GFP RNA T cells (GFP) were tested for their lytic activity using a cytotoxic T-lymphocyte assay at the effector:target ratio of 5:1 ( b ). The supernatant from the Blina-RNA T cells (Blina-RNA Sup.) was collected 18 h after electroporation, diluted 10 (1/10) or 100 times (1/100) with culture medium, added to T cells that were not electroporated with any RNA (No RNA) and co-cultured with the CD19 + cell lines (Nalm6, K562-CD19 or Raji cells). The K562 cell line was used as a negative control. T cells that had been electroporated with the CAR RNA or Blina-RNA were used as positive controls in the CD107a assay (gated on CD8 + T cells) ( c ) (representative of three independent experiments). * P <0.05; ** P <0.01.

Article Snippet: The Nalm6 (DSMZ, Braunschweig, Germany), Raji (American Type Culture Collection, Manassas, VA, USA) and K562 (American Type Culture Collection) cell lines were cultured per the providers' instructions.

Techniques: Electroporation, Staining, Expressing, Activity Assay, Cytotoxic T Lymphocyte Assay, Cell Culture, Negative Control

Journal: Blood Cancer Journal

Article Title: Improved anti-leukemia activities of adoptively transferred T cells expressing bispecific T-cell engager in mice

doi: 10.1038/bcj.2016.38

Figure Lengend Snippet: Blinatumomab BiTE RNA T cells were less dependent on co-stimulation and exhibited enhanced division and proliferation. CFSE-labeled resting CD4 T cells were electroporated with the blinatumomab BiTE RNA (Blina-RNA) or CD19-BBZ CAR RNA (CAR RNA) at the indicated RNA doses and stimulated with either irradiated K562-CD19 cells mixed with an equal amount of irradiated K562 cells (as control for K562-CD86; K-19/K562) or irradiated K562-CD19 cells mixed with an equal amount of irradiated K562-CD86 cells (K-19/K-86). The CFSE dilution was examined at day 6 (gated on CD3 + T cells). The results from a representative experiment are shown in a , and a summary of three independent experiments is shown in b . CFSE-labeled CD45RO + (memory) or CD45RO − (naive) resting CD4 T cells were electroporated with the blinatumomab BiTE RNA (Blina-RNA) or CD19-BBZ RNA (CAR RNA) at the indicated RNA doses and stimulated with either irradiated K562-CD19 cells mixed with an equal amount of irradiated K562 cells (as control for K562-CD86) or irradiated K562-CD19 cells mixed with an equal amount of irradiated K562-CD86 cells. The CFSE dilution was examined at day 6 (gated on CD3 + T cells) ( c ), and the T-cell expansion was monitored at different days after stimulation ( d ). The CSFE-labeled T cells that had been electroporated with either 1 or 5 μg of the CD19-BBZ (19BBZ), CD19-28Z (19-28Z) or blinatumomab BiTE (Blina-RNA) RNA were stimulated with irradiated K562, K562-CD19/K562 or K562-CD19/K562-CD86 cells. Six days later, the T cells were subjected to the flow cytometry analysis for CFSE dilution (gated on CD3 + T cells) ( e ). Five hundred thousand T cells that had been electroporated with 5 μg of the CD19-BBZ (19BBZ), CD19-28Z (19-28Z) or blinatumomab BiTE (Blina-RNA) RNA were stimulated with K562-CD19/K562 or K562-CD19/K562-CD86 cells. Six days later, the total number of viable T cells was counted, and the fold increase in T-cell expansion was calculated ( f ) (representative of two independent experiments). NS, not significant. * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: The Nalm6 (DSMZ, Braunschweig, Germany), Raji (American Type Culture Collection, Manassas, VA, USA) and K562 (American Type Culture Collection) cell lines were cultured per the providers' instructions.

Techniques: Labeling, Irradiation, Control, Flow Cytometry