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    Structured Review

    ATCC k562
    Evaluation of the genome-editing activity of nucleofected multi-NLS SpCas9 proteins. ( A , B ) Evaluation of the genome-editing activity of multi-NLS SpCas9 at the CCR5 site in <t>K562</t> ( A ) and Jurkat ( B ) cells. The results are quantified using T7E1 analysis and presented as mean ± SD ( n = 2). The difference between N0/C1 and multi-NLS constructs is analyzed by two-tailed Student’s t test. *, p
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    Images

    1) Product Images from "Systematic Investigation of the Effects of Multiple SV40 Nuclear Localization Signal Fusion on the Genome Editing Activity of Purified SpCas9"

    Article Title: Systematic Investigation of the Effects of Multiple SV40 Nuclear Localization Signal Fusion on the Genome Editing Activity of Purified SpCas9

    Journal: Bioengineering

    doi: 10.3390/bioengineering9020083

    Evaluation of the genome-editing activity of nucleofected multi-NLS SpCas9 proteins. ( A , B ) Evaluation of the genome-editing activity of multi-NLS SpCas9 at the CCR5 site in K562 ( A ) and Jurkat ( B ) cells. The results are quantified using T7E1 analysis and presented as mean ± SD ( n = 2). The difference between N0/C1 and multi-NLS constructs is analyzed by two-tailed Student’s t test. *, p
    Figure Legend Snippet: Evaluation of the genome-editing activity of nucleofected multi-NLS SpCas9 proteins. ( A , B ) Evaluation of the genome-editing activity of multi-NLS SpCas9 at the CCR5 site in K562 ( A ) and Jurkat ( B ) cells. The results are quantified using T7E1 analysis and presented as mean ± SD ( n = 2). The difference between N0/C1 and multi-NLS constructs is analyzed by two-tailed Student’s t test. *, p

    Techniques Used: Activity Assay, Construct, Two Tailed Test

    Evaluation of the effects of multi-NLS on SpCas9 activity and specificity in K562 cells. ( A ) The gene-editing activities of multi-NLS on SpCas9 at different dosage, as determined by T7E1 analysis. The data are shown as mean ± SD ( n = 3 technical replicates). ( B ) The effects of multi-NLS on the specificity of SpCas9 variants. The specificity is determined by the ratio between on-target and off-target activities. The difference between N0/C1 and multi-NLS constructs at each corresponding condition is analyzed by one-way ANOVA with Bonferroni’s multiple comparisons test. *, p
    Figure Legend Snippet: Evaluation of the effects of multi-NLS on SpCas9 activity and specificity in K562 cells. ( A ) The gene-editing activities of multi-NLS on SpCas9 at different dosage, as determined by T7E1 analysis. The data are shown as mean ± SD ( n = 3 technical replicates). ( B ) The effects of multi-NLS on the specificity of SpCas9 variants. The specificity is determined by the ratio between on-target and off-target activities. The difference between N0/C1 and multi-NLS constructs at each corresponding condition is analyzed by one-way ANOVA with Bonferroni’s multiple comparisons test. *, p

    Techniques Used: Activity Assay, Construct

    2) Product Images from "HMG2 Interacts with the Nucleosome Assembly Protein SET and Is a Target of the Cytotoxic T-Lymphocyte Protease Granzyme A"

    Article Title: HMG2 Interacts with the Nucleosome Assembly Protein SET and Is a Target of the Cytotoxic T-Lymphocyte Protease Granzyme A

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.8.2810-2820.2002

    HMG2 is cleaved in cells perforin loaded with GzmA. (a) K562 cells were treated for the indicated times at 37°C with 1 μM GzmA (A) or 1 μM S-AGzmA (S-A) and sublytic concentrations of perforin (P). Cell lysates were analyzed by immunoblotting for HMG2, SET, and pp32. Neither full-length HMG2 nor SET are detected after 2 h, but pp32 is unchanged. (b) Nuclear fractions were isolated 4 h after GzmA loading with perforin and analyzed by immunoblotting. Nuclear SET and HMG2 are completely degraded by active, but not inactive, enzyme in a perforin-dependent manner.
    Figure Legend Snippet: HMG2 is cleaved in cells perforin loaded with GzmA. (a) K562 cells were treated for the indicated times at 37°C with 1 μM GzmA (A) or 1 μM S-AGzmA (S-A) and sublytic concentrations of perforin (P). Cell lysates were analyzed by immunoblotting for HMG2, SET, and pp32. Neither full-length HMG2 nor SET are detected after 2 h, but pp32 is unchanged. (b) Nuclear fractions were isolated 4 h after GzmA loading with perforin and analyzed by immunoblotting. Nuclear SET and HMG2 are completely degraded by active, but not inactive, enzyme in a perforin-dependent manner.

    Techniques Used: Isolation

    GzmA, but not GzmB, cleaves native HMG2 in K562 cell lysates and isolated nuclei. (a) K562 postnuclear cytoplasmic lysates (2 × 10 5 equivalents) were incubated with the indicated concentrations of GzmA or 1 μM S-AGzmA or GzmB at 37°C for 2 h and analyzed by immunoblotting for HMG2, SET, and pp32. HMG2 and SET are cleaved at nanomolar concentrations of GzmA, but pp32 is unchanged. (b) Increasing concentrations of GzmA or 1 μM inactive S-AGzmA was incubated with 10 6 K562 nuclei and nuclear lysates were analyzed 1.5 h later for cleavage of HMG2, SET, pp32, and HMG1. Full-length SET and HMG2 are completely cleaved at nanomolar concentrations of GzmA.
    Figure Legend Snippet: GzmA, but not GzmB, cleaves native HMG2 in K562 cell lysates and isolated nuclei. (a) K562 postnuclear cytoplasmic lysates (2 × 10 5 equivalents) were incubated with the indicated concentrations of GzmA or 1 μM S-AGzmA or GzmB at 37°C for 2 h and analyzed by immunoblotting for HMG2, SET, and pp32. HMG2 and SET are cleaved at nanomolar concentrations of GzmA, but pp32 is unchanged. (b) Increasing concentrations of GzmA or 1 μM inactive S-AGzmA was incubated with 10 6 K562 nuclei and nuclear lysates were analyzed 1.5 h later for cleavage of HMG2, SET, pp32, and HMG1. Full-length SET and HMG2 are completely cleaved at nanomolar concentrations of GzmA.

    Techniques Used: Isolation, Incubation

    ). (b) rHMG2 binds directly to rSET. rHMG2 and rSET were coincubated and immunoprecipitated with anti-HMG2 antisera (left) or anti-SET monoclonal antibody (right) or control antibody. (c) The experiment shown in panel b was repeated with K562 postnuclear lysates to coprecipitate native SET and HMG2. (d) However, despite the close homology between HMG1 and HMG2, HMG1 and SET do not coprecipitate from K562 cell lysates. (e) rHMG2 does not bind directly to rAPE, another SET complex protein (Fan et al., submitted).
    Figure Legend Snippet: ). (b) rHMG2 binds directly to rSET. rHMG2 and rSET were coincubated and immunoprecipitated with anti-HMG2 antisera (left) or anti-SET monoclonal antibody (right) or control antibody. (c) The experiment shown in panel b was repeated with K562 postnuclear lysates to coprecipitate native SET and HMG2. (d) However, despite the close homology between HMG1 and HMG2, HMG1 and SET do not coprecipitate from K562 cell lysates. (e) rHMG2 does not bind directly to rAPE, another SET complex protein (Fan et al., submitted).

    Techniques Used: Immunoprecipitation

    HMG2 coelutes with the SET complex proteins SET and pp32. (a) A 270- to 420-kDa complex elutes from K562 cell lysates applied sequentially to immobilized S-AGzmA and S400 gel filtration columns. Migration of Pharmacia gel filtration standards is indicated. (b) The SET complex, isolated from the S-AGzmA affinity column, is disrupted by purification on an anion-exchange column. The acidic SET and pp32 proteins stick to the anion-exchange column (eluate, E), but most proteins are in the flowthrough (FT). HMG2 was identified by N-terminal sequencing of a prominent 28-kDa band in the flowthrough. The identification of the indicated bands was verified by immunoblotting (not shown). (c) Immunoblots confirm the comigration of HMG2, SET, and pp32 in the S400 column fractions.
    Figure Legend Snippet: HMG2 coelutes with the SET complex proteins SET and pp32. (a) A 270- to 420-kDa complex elutes from K562 cell lysates applied sequentially to immobilized S-AGzmA and S400 gel filtration columns. Migration of Pharmacia gel filtration standards is indicated. (b) The SET complex, isolated from the S-AGzmA affinity column, is disrupted by purification on an anion-exchange column. The acidic SET and pp32 proteins stick to the anion-exchange column (eluate, E), but most proteins are in the flowthrough (FT). HMG2 was identified by N-terminal sequencing of a prominent 28-kDa band in the flowthrough. The identification of the indicated bands was verified by immunoblotting (not shown). (c) Immunoblots confirm the comigration of HMG2, SET, and pp32 in the S400 column fractions.

    Techniques Used: Filtration, Migration, Isolation, Affinity Column, Purification, Sequencing, Western Blot

    3) Product Images from "A Recurring Chemogenetic Switch for Chimeric Antigen Receptor T Cells"

    Article Title: A Recurring Chemogenetic Switch for Chimeric Antigen Receptor T Cells

    Journal: bioRxiv

    doi: 10.1101/2021.08.23.457355

    sCAR19 T cells exhibit cytotoxicity in vitro . (a) Cytotoxicity of un-transduced T cells, CAR19 T cells and sCAR19 T cells cultured in two conditions, one with the DMSO vehicle and the other with 1 μM ASV toward CD19 + Raji (the left graph) and CD19 - K562 cells (the right graph). Both Raji and K562 cells were labeled with calcein-AM before they were cocultured with four groups of T cells with ratios of effector to target tumor cells (E:T) as indicated in the figures for 4 h. Lysis of target cells was analyzed by detecting released calcein-AM in media. Data are representative of three independent experiments and normalized against total lysis of calcein-AM-labeled Raji and K562 cells. (b) The release of cytokines including IFN-γ, IL-2 and TNFα from un-transduced T cells, CAR19 T cells and sCAR19 T cells cultured in two conditions, one with the DMSO vehicle and the other with 1 μM ASV when they were cocultured with Raji cells with a E:T ratio as 1:1 for 24 h. Cytokine levels were detected using ELISA.
    Figure Legend Snippet: sCAR19 T cells exhibit cytotoxicity in vitro . (a) Cytotoxicity of un-transduced T cells, CAR19 T cells and sCAR19 T cells cultured in two conditions, one with the DMSO vehicle and the other with 1 μM ASV toward CD19 + Raji (the left graph) and CD19 - K562 cells (the right graph). Both Raji and K562 cells were labeled with calcein-AM before they were cocultured with four groups of T cells with ratios of effector to target tumor cells (E:T) as indicated in the figures for 4 h. Lysis of target cells was analyzed by detecting released calcein-AM in media. Data are representative of three independent experiments and normalized against total lysis of calcein-AM-labeled Raji and K562 cells. (b) The release of cytokines including IFN-γ, IL-2 and TNFα from un-transduced T cells, CAR19 T cells and sCAR19 T cells cultured in two conditions, one with the DMSO vehicle and the other with 1 μM ASV when they were cocultured with Raji cells with a E:T ratio as 1:1 for 24 h. Cytokine levels were detected using ELISA.

    Techniques Used: In Vitro, Cell Culture, Labeling, Lysis, Enzyme-linked Immunosorbent Assay

    4) Product Images from "AmpliconReconstructor integrates NGS and optical mapping to resolve the complex structures of focal amplifications"

    Article Title: AmpliconReconstructor integrates NGS and optical mapping to resolve the complex structures of focal amplifications

    Journal: Nature Communications

    doi: 10.1038/s41467-020-18099-z

    Reconstruction of a complex Philadelphia chromosome. a AA-generated breakpoint graph for K562. Estimated copy number (CN), coverage, discordant reads forming breakpoint graph edges, and a subset of the genes in these regions are shown. b AR reconstruction of an 8.5 Mbp focal amplification which was supported by both Irys and Saphyr reconstructions. The tracks from top to bottom are: OM contigs (with contig ID and direction indicated above), graph segments (alignments shown with vertical gray lines), gene subset, and color-coded reference genome bar with genomic coordinates (scaled as 10 kbp units). Gray half-height bars between individual segments on the reference genome bar indicate support from edges in the AA breakpoint graph. White arrows inside the chromosome color bar indicate direction of genomic segment(s). Colored numbers correspond to numbered breakpoint graph edges in panel ( a ). c Multi-FISH using probes against BCR , ABL1 , and GPC5 with DAPI-stained metaphase chromosomes. Scale bars indicate 2 µm in both “Full size” and “Zoom” rows.
    Figure Legend Snippet: Reconstruction of a complex Philadelphia chromosome. a AA-generated breakpoint graph for K562. Estimated copy number (CN), coverage, discordant reads forming breakpoint graph edges, and a subset of the genes in these regions are shown. b AR reconstruction of an 8.5 Mbp focal amplification which was supported by both Irys and Saphyr reconstructions. The tracks from top to bottom are: OM contigs (with contig ID and direction indicated above), graph segments (alignments shown with vertical gray lines), gene subset, and color-coded reference genome bar with genomic coordinates (scaled as 10 kbp units). Gray half-height bars between individual segments on the reference genome bar indicate support from edges in the AA breakpoint graph. White arrows inside the chromosome color bar indicate direction of genomic segment(s). Colored numbers correspond to numbered breakpoint graph edges in panel ( a ). c Multi-FISH using probes against BCR , ABL1 , and GPC5 with DAPI-stained metaphase chromosomes. Scale bars indicate 2 µm in both “Full size” and “Zoom” rows.

    Techniques Used: Generated, Amplification, Fluorescence In Situ Hybridization, Staining

    5) Product Images from "Sleeping Beauty system to redirect T-cell specificity for human applications"

    Article Title: Sleeping Beauty system to redirect T-cell specificity for human applications

    Journal: Journal of immunotherapy (Hagerstown, Md. : 1997)

    doi: 10.1097/CJI.0b013e3182811ce9

    K562-derived aAPC (clone #4) sustains proliferation of CAR + T cells
    Figure Legend Snippet: K562-derived aAPC (clone #4) sustains proliferation of CAR + T cells

    Techniques Used: Derivative Assay

    6) Product Images from "Characterization of New Syncytium-Inhibiting Monoclonal Antibodies Implicates Lipid Rafts in Human T-Cell Leukemia Virus Type 1 Syncytium Formation"

    Article Title: Characterization of New Syncytium-Inhibiting Monoclonal Antibodies Implicates Lipid Rafts in Human T-Cell Leukemia Virus Type 1 Syncytium Formation

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.16.7351-7361.2001

    BCD does not alter VCAM-1 binding. K562/VCAM cells were pretreated with BCD, followed by preincubation with anti-VCAM-1 MAb VIII6G10 or media alone. They were then incubated with Jurkat T cells for 1 h at 37°C as described in Materials and Methods. Conjugate formation was visualized by photomicroscopy 1 h after mixing the cells. Top row, left to right: untreated Jurkat cells and untreated K562/VCAM1 cells alone, mixed with Jurkat cells, and mixed with Jurkat cells in the presence of anti-VCAM1 MAb (with anti-VCAM1). Bottom row, left to right: BCD-treated K562/VCAM1 cells alone, mixed with Jurkat cells (middle panel), and mixed with Jurkat cells in the presence of anti-VCAM1 MAb (right panel).
    Figure Legend Snippet: BCD does not alter VCAM-1 binding. K562/VCAM cells were pretreated with BCD, followed by preincubation with anti-VCAM-1 MAb VIII6G10 or media alone. They were then incubated with Jurkat T cells for 1 h at 37°C as described in Materials and Methods. Conjugate formation was visualized by photomicroscopy 1 h after mixing the cells. Top row, left to right: untreated Jurkat cells and untreated K562/VCAM1 cells alone, mixed with Jurkat cells, and mixed with Jurkat cells in the presence of anti-VCAM1 MAb (with anti-VCAM1). Bottom row, left to right: BCD-treated K562/VCAM1 cells alone, mixed with Jurkat cells (middle panel), and mixed with Jurkat cells in the presence of anti-VCAM1 MAb (right panel).

    Techniques Used: Binding Assay, Incubation

    New MAbs against K562 cells block HTLV-1 syncytium formation. (A and B) MAbs produced against K562 cells (K5.M series) were tested for inhibition of HTLV-1-induced syncytium formation between K562/VCAM cells and MT2 cells, as described in Materials and Methods. Myeloma IgG2b and anti-VCAM-1 (VIII6G10) antibodies were used as negative and positive controls, respectively. (B) Data shown are mean numbers of syncytia per HPF. Syncytia in six HPFs were counted. (C) MAbs were tested for inhibition of HTLV-1-induced syncytium formation between K562/VCAM cells and MJG11 cells.
    Figure Legend Snippet: New MAbs against K562 cells block HTLV-1 syncytium formation. (A and B) MAbs produced against K562 cells (K5.M series) were tested for inhibition of HTLV-1-induced syncytium formation between K562/VCAM cells and MT2 cells, as described in Materials and Methods. Myeloma IgG2b and anti-VCAM-1 (VIII6G10) antibodies were used as negative and positive controls, respectively. (B) Data shown are mean numbers of syncytia per HPF. Syncytia in six HPFs were counted. (C) MAbs were tested for inhibition of HTLV-1-induced syncytium formation between K562/VCAM cells and MJG11 cells.

    Techniques Used: Blocking Assay, Produced, Inhibition

    Immunoblot of K562 cell fractions shows that proteins recognized by K5.M MAbs are in lipid rafts. Nonionic detergent lysates were prepared from K562 cells and subjected to equilibrium centrifugation on sucrose gradients, and fractions were collected as described in Materials and Methods. The fractions were subjected to immunoblot analysis with the MAbs indicated. Lipid raft and soluble fractions were identified by anti-CD59 and anti-CD71 MAbs, respectively. Anti-MHC-1 MAb (MHM.5) served as a negative control.
    Figure Legend Snippet: Immunoblot of K562 cell fractions shows that proteins recognized by K5.M MAbs are in lipid rafts. Nonionic detergent lysates were prepared from K562 cells and subjected to equilibrium centrifugation on sucrose gradients, and fractions were collected as described in Materials and Methods. The fractions were subjected to immunoblot analysis with the MAbs indicated. Lipid raft and soluble fractions were identified by anti-CD59 and anti-CD71 MAbs, respectively. Anti-MHC-1 MAb (MHM.5) served as a negative control.

    Techniques Used: Centrifugation, Negative Control

    7) Product Images from "Potent and selective antitumor activity of a T cell-engaging bispecific antibody targeting a membrane-proximal epitope of ROR1"

    Article Title: Potent and selective antitumor activity of a T cell-engaging bispecific antibody targeting a membrane-proximal epitope of ROR1

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1719905115

    Cell surface binding of bispecific ROR1 × CD3 scFv-Fc. The indicated bispecific ROR1 × CD3 biAbs were analyzed for binding to human CD3-positive T cell line Jurkat and human ROR1-positive cell lines K562/ROR1 and JeKo-1 by flow cytometry at a concentration of 5 μg/mL followed by APC-conjugated goat anti-human IgG pAbs. Parental K562 is a ROR1-negative control cell line. Open histograms show the background binding signal of the secondary pAbs.
    Figure Legend Snippet: Cell surface binding of bispecific ROR1 × CD3 scFv-Fc. The indicated bispecific ROR1 × CD3 biAbs were analyzed for binding to human CD3-positive T cell line Jurkat and human ROR1-positive cell lines K562/ROR1 and JeKo-1 by flow cytometry at a concentration of 5 μg/mL followed by APC-conjugated goat anti-human IgG pAbs. Parental K562 is a ROR1-negative control cell line. Open histograms show the background binding signal of the secondary pAbs.

    Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Concentration Assay, Negative Control

    T cell activation mediated by bispecific ROR1 × CD3 scFv-Fc. Ex vivo expanded primary human T cells were incubated with 1 μg/mL of the indicated biAbs in the presence of K562/ROR1 or K562 cells at an effector-to-target ratio of 10:1 for 16 h. ( A ) Percentage of activated T cells based on CD69 expression. Cytokine release measured by ELISA for IFN-γ ( B ), TNF-α ( C ), and IL-2 ( D ). Shown are mean ± SD values for independent triplicates. One-way ANOVA was used to analyze significant differences between ROR1 × CD3 (colored) and control scFv-Fc (black; * P
    Figure Legend Snippet: T cell activation mediated by bispecific ROR1 × CD3 scFv-Fc. Ex vivo expanded primary human T cells were incubated with 1 μg/mL of the indicated biAbs in the presence of K562/ROR1 or K562 cells at an effector-to-target ratio of 10:1 for 16 h. ( A ) Percentage of activated T cells based on CD69 expression. Cytokine release measured by ELISA for IFN-γ ( B ), TNF-α ( C ), and IL-2 ( D ). Shown are mean ± SD values for independent triplicates. One-way ANOVA was used to analyze significant differences between ROR1 × CD3 (colored) and control scFv-Fc (black; * P

    Techniques Used: Activation Assay, Ex Vivo, Incubation, Expressing, Enzyme-linked Immunosorbent Assay

    T cell engagement mediated by bispecific ROR1 × CD3 scFv-Fc. ( A ) Cross-linking of 5 × 10 4 primary human T cells (stained with CellTrace Far Red) and 5 × 10 4 K562/ROR1 cells (stained with CellTrace CFSE) in the presence of 1 µg/mL ROR1 × CD3 and control scFv-Fc. Double-stained events were detected by flow cytometry. ( B ) Quantification of double-stained events from three independent triplicates (mean ± SD). ( C ) The cytotoxicity of ROR1 × CD3 scFv-Fc was tested with ex vivo expanded primary human T cells and K562/ROR1 ( C ), JeKo-1 ( D ), or K562 ( E ) cells at an effector-to-target ratio of 10:1 and measured after 16 h. Shown are mean ± SD values from three independent triplicates.
    Figure Legend Snippet: T cell engagement mediated by bispecific ROR1 × CD3 scFv-Fc. ( A ) Cross-linking of 5 × 10 4 primary human T cells (stained with CellTrace Far Red) and 5 × 10 4 K562/ROR1 cells (stained with CellTrace CFSE) in the presence of 1 µg/mL ROR1 × CD3 and control scFv-Fc. Double-stained events were detected by flow cytometry. ( B ) Quantification of double-stained events from three independent triplicates (mean ± SD). ( C ) The cytotoxicity of ROR1 × CD3 scFv-Fc was tested with ex vivo expanded primary human T cells and K562/ROR1 ( C ), JeKo-1 ( D ), or K562 ( E ) cells at an effector-to-target ratio of 10:1 and measured after 16 h. Shown are mean ± SD values from three independent triplicates.

    Techniques Used: Staining, Flow Cytometry, Cytometry, Ex Vivo

    8) Product Images from "Human Cancer-Associated Mutations of SF3B1 Lead to a Splicing Modification of Its Own RNA"

    Article Title: Human Cancer-Associated Mutations of SF3B1 Lead to a Splicing Modification of Its Own RNA

    Journal: Cancers

    doi: 10.3390/cancers12030652

    SF3B1ins protein is defective for splicing. ( A – E ) K562 cells were transfected with plasmids encoding different versions of SF3B1 (SF3B1 WT , SF3B1 K700E , SF3B1 WT ins, and SF3B1 K700E ins). ( A ) Total SF3B1 proteins (endogenous and exogenous) were detected by Western blot analysis using anti-SF3B1 antibody. Plasmid-encoded SF3B1 was detected using anti-FLAG antibody. ( B ) Analysis of splicing events known to be specifically altered in SF3B1 -mutated background (ENOSF1, TMEM14C, DPH5) or upon SF3B1 loss of function (RBM5, DUSP11). RT-PCR was performed using primers allowing specific detection of distinct splicing events in SF3B1, ENOSF1, TMEM14C, DPH5, RBM5, and DUSP11. ( C – E ) K562 cells were co-transfected with an siRNA specific to endogenous SF3B1 and with plasmids encoding different versions of SF3B1. ( C ) Western blot analysis of SF3B1 proteins as described in (A). Average quantification of endogenous and exogenous levels of SF3B1 protein from three independent experiments is indicated. ( D ) Analysis of specific splicing events as described in ( B ). The proportion of exon skipping in RBM5 and DUSP11 (average from three independent experiments) is indicated below the corresponding gels. ( E ) RT-qPCR was performed using primers allowing specific quantification of exon skipping events in CCNA2 (exon 5) and STK6 (exons 4, 5, and 6) transcripts, normalized to GAPDH. Relative quantification is indicated, and error bars represent ± SEM from three independent experiments. ( F ) Growth of K562 cells expressing inducible SF3B1 WT versus SF3B1 WT ins (top) and SF3B1 K700E versus SF3B1 K700E ins (bottom) upon SF3B1 silencing (shSF3B1), following doxycycline induction. Error bars represent ± SEM from four independent experiments. A Mann–Whitney test was applied ( p value
    Figure Legend Snippet: SF3B1ins protein is defective for splicing. ( A – E ) K562 cells were transfected with plasmids encoding different versions of SF3B1 (SF3B1 WT , SF3B1 K700E , SF3B1 WT ins, and SF3B1 K700E ins). ( A ) Total SF3B1 proteins (endogenous and exogenous) were detected by Western blot analysis using anti-SF3B1 antibody. Plasmid-encoded SF3B1 was detected using anti-FLAG antibody. ( B ) Analysis of splicing events known to be specifically altered in SF3B1 -mutated background (ENOSF1, TMEM14C, DPH5) or upon SF3B1 loss of function (RBM5, DUSP11). RT-PCR was performed using primers allowing specific detection of distinct splicing events in SF3B1, ENOSF1, TMEM14C, DPH5, RBM5, and DUSP11. ( C – E ) K562 cells were co-transfected with an siRNA specific to endogenous SF3B1 and with plasmids encoding different versions of SF3B1. ( C ) Western blot analysis of SF3B1 proteins as described in (A). Average quantification of endogenous and exogenous levels of SF3B1 protein from three independent experiments is indicated. ( D ) Analysis of specific splicing events as described in ( B ). The proportion of exon skipping in RBM5 and DUSP11 (average from three independent experiments) is indicated below the corresponding gels. ( E ) RT-qPCR was performed using primers allowing specific quantification of exon skipping events in CCNA2 (exon 5) and STK6 (exons 4, 5, and 6) transcripts, normalized to GAPDH. Relative quantification is indicated, and error bars represent ± SEM from three independent experiments. ( F ) Growth of K562 cells expressing inducible SF3B1 WT versus SF3B1 WT ins (top) and SF3B1 K700E versus SF3B1 K700E ins (bottom) upon SF3B1 silencing (shSF3B1), following doxycycline induction. Error bars represent ± SEM from four independent experiments. A Mann–Whitney test was applied ( p value

    Techniques Used: Transfection, Western Blot, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, MANN-WHITNEY

    A novel splicing isoform of SF3B1 , SF3B1ins, is detected in sideroblastic myelodysplastic syndromes (MDS-RS) patients and myeloid cell lines expressing mutant SF3B1 . ( A ) Representation of the aberrantly spliced junction of SF3B1 . ( B ) 3D modeling of the eight amino acid insertion in the H3 repeat of SF3B1 Huntingtin, Elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1 (HEAT) domain. The site of insertion and the lysine in position 700 are indicated in both normal (top) and predicted (bottom) structures. Primary sequences of normal and aberrant H3 repeat (amino acids 588–605) are indicated below the 3D structures. The inserted sequence is colored in orange. ( C ) Detection of SF3B1ins transcript in mononuclear cells from myelodysplastic syndromes (MDS) patients harboring SF3B1 mutations. RNA was extracted from mononuclear cells of subjects with normal bone marrow (lanes 1–4), from MDS patients without RS (lanes 5–10) and from MDS-RS patients. The mutational status of SF3B1 is indicated for MDS-RS patients, as follows: mut—mutated; WT—wild-type. RT-PCR was performed using primers allowing specific detection of SF3B1ins or detection of both aberrant (upper band) and canonical (lower band) transcripts of SF3B1, ENOSF1 and TMEM14C. (°: ENOSF1 RT-PCR on patient 2 could not be performed due to an insufficient quantity of material); ( D , E ) Inducible expression of SF3B1 K700E in K562 cells and UT-7 cells generates SF3B1ins transcript. RT-PCR was performed from K562 cells ( D ) and UT-7 ( E ), expressing SF3B1 WT (left) or SF3B1 K700E (right) (I: induced; NI: non induced), using primers as described in C. Steady-state SF3B1 protein level was achieved by Western blot ( Figure S1 ); ( F ) Detection of SF3B1ins transcript in K562 transiently expressing distinct SF3B1 variants. RT-PCR was performed using primers as described in C. Data information: In ( D , E ), representative results of at least three independent RT-PCR experiments are presented. L: ladder.
    Figure Legend Snippet: A novel splicing isoform of SF3B1 , SF3B1ins, is detected in sideroblastic myelodysplastic syndromes (MDS-RS) patients and myeloid cell lines expressing mutant SF3B1 . ( A ) Representation of the aberrantly spliced junction of SF3B1 . ( B ) 3D modeling of the eight amino acid insertion in the H3 repeat of SF3B1 Huntingtin, Elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1 (HEAT) domain. The site of insertion and the lysine in position 700 are indicated in both normal (top) and predicted (bottom) structures. Primary sequences of normal and aberrant H3 repeat (amino acids 588–605) are indicated below the 3D structures. The inserted sequence is colored in orange. ( C ) Detection of SF3B1ins transcript in mononuclear cells from myelodysplastic syndromes (MDS) patients harboring SF3B1 mutations. RNA was extracted from mononuclear cells of subjects with normal bone marrow (lanes 1–4), from MDS patients without RS (lanes 5–10) and from MDS-RS patients. The mutational status of SF3B1 is indicated for MDS-RS patients, as follows: mut—mutated; WT—wild-type. RT-PCR was performed using primers allowing specific detection of SF3B1ins or detection of both aberrant (upper band) and canonical (lower band) transcripts of SF3B1, ENOSF1 and TMEM14C. (°: ENOSF1 RT-PCR on patient 2 could not be performed due to an insufficient quantity of material); ( D , E ) Inducible expression of SF3B1 K700E in K562 cells and UT-7 cells generates SF3B1ins transcript. RT-PCR was performed from K562 cells ( D ) and UT-7 ( E ), expressing SF3B1 WT (left) or SF3B1 K700E (right) (I: induced; NI: non induced), using primers as described in C. Steady-state SF3B1 protein level was achieved by Western blot ( Figure S1 ); ( F ) Detection of SF3B1ins transcript in K562 transiently expressing distinct SF3B1 variants. RT-PCR was performed using primers as described in C. Data information: In ( D , E ), representative results of at least three independent RT-PCR experiments are presented. L: ladder.

    Techniques Used: Expressing, Mutagenesis, Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot

    9) Product Images from "Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting"

    Article Title: Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1136/jitc-2020-001334

    NK cells cluster and polarize lytic granules after contact with the susceptible cell line K562. (A) Lytic granules of NK-92 were stained with Lysotracker (red), and live-cell imaging of NK-92 cocultured with susceptible K562 cells was performed. White arrows show conjugates, and blue arrows show dead cells. Representative images are shown. Time in hh:mm. (B) Granules were further segmented using Imaris software for quantification. (C) Lytic granule clustering was measured as the mean distance of single granules to the granules cluster center (replacing the microtubule-organizing center). (D) Granule polarization was measured as the mean distance of single lytic granules to the immunological synapse (IS), identified as the conjugation point of NK and target cells in 3D reconstructed images. Granule clustering and polarization in each cell was followed and analyzed before (pre) and after (post) stimulation with target cells. Only cytotoxcic NK cells that formed conjugates were counted. Each dot represents the mean of granules per cell per experiment. Data represent at least 3 independent experiments. 3D, 3 dimensional; NK, natural killer. ***P
    Figure Legend Snippet: NK cells cluster and polarize lytic granules after contact with the susceptible cell line K562. (A) Lytic granules of NK-92 were stained with Lysotracker (red), and live-cell imaging of NK-92 cocultured with susceptible K562 cells was performed. White arrows show conjugates, and blue arrows show dead cells. Representative images are shown. Time in hh:mm. (B) Granules were further segmented using Imaris software for quantification. (C) Lytic granule clustering was measured as the mean distance of single granules to the granules cluster center (replacing the microtubule-organizing center). (D) Granule polarization was measured as the mean distance of single lytic granules to the immunological synapse (IS), identified as the conjugation point of NK and target cells in 3D reconstructed images. Granule clustering and polarization in each cell was followed and analyzed before (pre) and after (post) stimulation with target cells. Only cytotoxcic NK cells that formed conjugates were counted. Each dot represents the mean of granules per cell per experiment. Data represent at least 3 independent experiments. 3D, 3 dimensional; NK, natural killer. ***P

    Techniques Used: Staining, Live Cell Imaging, Software, Conjugation Assay

    Both CAR-retargeted and FcR-mAb-retargeted NK cells overcome cancer cell resistance. (A) Flow cytometry analysis of ErbB2 expression in MDA-MB-453 and K562 cancer cell lines. isotype controls are shown in gray. (B) CAR expression on NK-92/5.28.z (left) was detected by ErbB2-Fc combined with anti-Fc secondary antibody. FcR on haNK (right) was stained with anti-CD16 antibody. Dashed lines indicate stained parental NK-92 cells, and filled gray areas indicate unstained controls. Representative data from at least 3 independent experiments are shown. (C) Specific cytotoxicity of NK-92/5.28.z and haNK+Herceptin toward MDA-MB-453 was measured by Europium-based assay after 2 h co-culture. NK-92 and haNK without Herceptin were used as negative controls. (D) Cytotoxicity of NK-92/5.28.z and haNK+Herceptin toward MDA-MB-453 was monitored by live-cell imaging (SYTOX uptake) over time. Statistical analysis is shown for the 4 hour and 9 hours coincubation time points. Data represent at least 3 independent experiments. Mean values ±SEM are shown. n=25 (NK-92/5.28.z) and 15 (haNK) conjugates. (E) Time-lapse imaging of NK-92/5.28.z with MDA-MB-453. (a) Unconjugated NK cell with dispersed lytic granules. (b–e) An NK-92/5.28.z cell binds to two target cells, clusters and polarizes its lytic granules (white arrows) and (d–f) kills the cancer cells (blue arrows). (F) Time-lapse imaging of haNK+Herceptin with MDA-MB-453. (A) Two haNK cells form a conjugate with a target cell. (B–E) Granule clustering and polarization of both haNK cells occurs toward the conjugation points (white arrows). (D–F) The target cell turns blue, indicating cell death (blue arrows). Time in hh:mm. Representative images of at least four independent experiments are shown. CAR, chimeric antigen receptor; FcR, Fc receptors; haNK, high-affinity natural killer; ns, not significant; ****P
    Figure Legend Snippet: Both CAR-retargeted and FcR-mAb-retargeted NK cells overcome cancer cell resistance. (A) Flow cytometry analysis of ErbB2 expression in MDA-MB-453 and K562 cancer cell lines. isotype controls are shown in gray. (B) CAR expression on NK-92/5.28.z (left) was detected by ErbB2-Fc combined with anti-Fc secondary antibody. FcR on haNK (right) was stained with anti-CD16 antibody. Dashed lines indicate stained parental NK-92 cells, and filled gray areas indicate unstained controls. Representative data from at least 3 independent experiments are shown. (C) Specific cytotoxicity of NK-92/5.28.z and haNK+Herceptin toward MDA-MB-453 was measured by Europium-based assay after 2 h co-culture. NK-92 and haNK without Herceptin were used as negative controls. (D) Cytotoxicity of NK-92/5.28.z and haNK+Herceptin toward MDA-MB-453 was monitored by live-cell imaging (SYTOX uptake) over time. Statistical analysis is shown for the 4 hour and 9 hours coincubation time points. Data represent at least 3 independent experiments. Mean values ±SEM are shown. n=25 (NK-92/5.28.z) and 15 (haNK) conjugates. (E) Time-lapse imaging of NK-92/5.28.z with MDA-MB-453. (a) Unconjugated NK cell with dispersed lytic granules. (b–e) An NK-92/5.28.z cell binds to two target cells, clusters and polarizes its lytic granules (white arrows) and (d–f) kills the cancer cells (blue arrows). (F) Time-lapse imaging of haNK+Herceptin with MDA-MB-453. (A) Two haNK cells form a conjugate with a target cell. (B–E) Granule clustering and polarization of both haNK cells occurs toward the conjugation points (white arrows). (D–F) The target cell turns blue, indicating cell death (blue arrows). Time in hh:mm. Representative images of at least four independent experiments are shown. CAR, chimeric antigen receptor; FcR, Fc receptors; haNK, high-affinity natural killer; ns, not significant; ****P

    Techniques Used: Flow Cytometry, Expressing, Multiple Displacement Amplification, Staining, Co-Culture Assay, Live Cell Imaging, Imaging, Conjugation Assay

    CAR and FcR-mAb retargeted NK cells remain in permanent target cell contact and their different dynamics of steps involved in cytotoxicity correlate with cell killing. Conjugation of NK cells with their target was monitored over time by live-cell imaging. (A) Target cells in stable conjugates (over 15-min duration) as percentage of all target cells. n=6 experiments for NK-92/5.28.z (48 targets), 4 experiments for haNK (32 targets). (B) Duration of MDA-MB-453 conjugations with NK-92 or retargeted variants. (C) Percentage of all detached NK cells from the MDA-MB-453 cell line. Data represent at least three independent experiments. mean values ±SD are shown. NK-92, NK-92/5.28.z and haNK were cocultured with susceptible K562 or resistant MDA-MB-453 targets and analyzed by live-cell imaging. (D) Number of contacts required by a single NK cell to successfully kill the given target. (E) Target cells (TC) that formed conjugates with NK cells were analyzed for sytox uptake as a marker for cell death. (F, G) Time period between conjugation and granule polarization (F) or polarization and target cell death (G). (H) Duration of the complete killing process, starting with NK and target cell conjugation, and ending with sytox uptake. The time period until polarization is marked. Data are pooled from at least three independent experiments. Mean values ±SEM are shown. CAR, chimeric antigen receptor; FcR, Fc receptors; haNK, high-affinity NK; NK, natural killer; ns, not significant; *P
    Figure Legend Snippet: CAR and FcR-mAb retargeted NK cells remain in permanent target cell contact and their different dynamics of steps involved in cytotoxicity correlate with cell killing. Conjugation of NK cells with their target was monitored over time by live-cell imaging. (A) Target cells in stable conjugates (over 15-min duration) as percentage of all target cells. n=6 experiments for NK-92/5.28.z (48 targets), 4 experiments for haNK (32 targets). (B) Duration of MDA-MB-453 conjugations with NK-92 or retargeted variants. (C) Percentage of all detached NK cells from the MDA-MB-453 cell line. Data represent at least three independent experiments. mean values ±SD are shown. NK-92, NK-92/5.28.z and haNK were cocultured with susceptible K562 or resistant MDA-MB-453 targets and analyzed by live-cell imaging. (D) Number of contacts required by a single NK cell to successfully kill the given target. (E) Target cells (TC) that formed conjugates with NK cells were analyzed for sytox uptake as a marker for cell death. (F, G) Time period between conjugation and granule polarization (F) or polarization and target cell death (G). (H) Duration of the complete killing process, starting with NK and target cell conjugation, and ending with sytox uptake. The time period until polarization is marked. Data are pooled from at least three independent experiments. Mean values ±SEM are shown. CAR, chimeric antigen receptor; FcR, Fc receptors; haNK, high-affinity NK; NK, natural killer; ns, not significant; *P

    Techniques Used: Conjugation Assay, Live Cell Imaging, Multiple Displacement Amplification, Marker

    NK cells form stable conjugates with resistant tumor cells. (A) Live-cell confocal microscopy of NK-92 and K562 cells. Target cells are stained with CellMask (green), and dead cells visualized by sytox reagent (blue). Single channels for transmitted light, each fluorescent channel and a merge of all channels are shown for two time points: early conjugation (upper row, white arrows show conjugates) and after target cell death (bottom row, the blue arrow shows a dead target). (B) Time-lapse imaging of NK-92 interaction with susceptible K562 cells (upper image sequence) or the resistant breast cancer cell line MDA-MB-453 (453; bottom image sequence). Conjugations are indicated by white arrows, and target cell death by blue arrows. Time in hh:mm. (C) Sytox uptake of target cells in conjugates over time is shown as the mean fluorescence intensity (MFI) relative to maximum MFI. Data from at least 3 independent experiments done on separate days are shown (13 conjugates for K562 and 19 conjugates for MDA-MB-453 were analyzed). (D) NK-92 and target cells were cocultured at the given E/T ratios for 2 hours, and specific cytotoxicity was measured by Europium-based cytotoxicity assay. Mean values ±SEM are shown. (E) Conjugate formation was monitored during live-cell imaging. Target cells (TC) stably conjugated to NK cells are plotted as a percentage of all observed target cells (n=26 for K562, n=21 for MDA-MB-453). Data represent at least 3 independent experiments done on separate days. Mean values ±SD are shown. NK, natural killer.
    Figure Legend Snippet: NK cells form stable conjugates with resistant tumor cells. (A) Live-cell confocal microscopy of NK-92 and K562 cells. Target cells are stained with CellMask (green), and dead cells visualized by sytox reagent (blue). Single channels for transmitted light, each fluorescent channel and a merge of all channels are shown for two time points: early conjugation (upper row, white arrows show conjugates) and after target cell death (bottom row, the blue arrow shows a dead target). (B) Time-lapse imaging of NK-92 interaction with susceptible K562 cells (upper image sequence) or the resistant breast cancer cell line MDA-MB-453 (453; bottom image sequence). Conjugations are indicated by white arrows, and target cell death by blue arrows. Time in hh:mm. (C) Sytox uptake of target cells in conjugates over time is shown as the mean fluorescence intensity (MFI) relative to maximum MFI. Data from at least 3 independent experiments done on separate days are shown (13 conjugates for K562 and 19 conjugates for MDA-MB-453 were analyzed). (D) NK-92 and target cells were cocultured at the given E/T ratios for 2 hours, and specific cytotoxicity was measured by Europium-based cytotoxicity assay. Mean values ±SEM are shown. (E) Conjugate formation was monitored during live-cell imaging. Target cells (TC) stably conjugated to NK cells are plotted as a percentage of all observed target cells (n=26 for K562, n=21 for MDA-MB-453). Data represent at least 3 independent experiments done on separate days. Mean values ±SD are shown. NK, natural killer.

    Techniques Used: Confocal Microscopy, Staining, Conjugation Assay, Imaging, Sequencing, Multiple Displacement Amplification, Fluorescence, Cytotoxicity Assay, Live Cell Imaging, Stable Transfection

    10) Product Images from "2B4 (CD244) signaling by recombinant antigen-specific chimeric receptors costimulates natural killer cell activation to leukemia and neuroblastoma cells"

    Article Title: 2B4 (CD244) signaling by recombinant antigen-specific chimeric receptors costimulates natural killer cell activation to leukemia and neuroblastoma cells

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-08-2810

    CAR transduced NK cells functionally interact with antigen-expressing tumor targets (A) Expression of the 2B4 ligand CD48 on the leukemia target cell lines (dashed line, isotype control; solid line, antibody). (B) CD25 upregulation and (C) intracellular interferon (IFN)-γ and tumor necrosis factor (TNF)-α expression by non-transduced (NT), CD19-ζ, CD19-2B4, CD19-t2B4, CD19-2B4ζ, CD19-t2B4ζ, and (B) CD19-41BBζ transduced NK cells were quantified by flow cytometry after 24-hour (B) or 6-hour (C) coincubation with CD19+ REH or SupB15 leukemia cells, CD19-negative ML-2 leukemia cells, K562, or medium alone, as indicated. To exclude non-NK lymphocytes and nontransduced NK cells within the cultures from analysis, the gate was set on GFP/CD56-coexpressing cells. Shown is one representative experiment of three. *p
    Figure Legend Snippet: CAR transduced NK cells functionally interact with antigen-expressing tumor targets (A) Expression of the 2B4 ligand CD48 on the leukemia target cell lines (dashed line, isotype control; solid line, antibody). (B) CD25 upregulation and (C) intracellular interferon (IFN)-γ and tumor necrosis factor (TNF)-α expression by non-transduced (NT), CD19-ζ, CD19-2B4, CD19-t2B4, CD19-2B4ζ, CD19-t2B4ζ, and (B) CD19-41BBζ transduced NK cells were quantified by flow cytometry after 24-hour (B) or 6-hour (C) coincubation with CD19+ REH or SupB15 leukemia cells, CD19-negative ML-2 leukemia cells, K562, or medium alone, as indicated. To exclude non-NK lymphocytes and nontransduced NK cells within the cultures from analysis, the gate was set on GFP/CD56-coexpressing cells. Shown is one representative experiment of three. *p

    Techniques Used: Expressing, Flow Cytometry, Cytometry

    CAR transduced NK cells show powerful cytolytic responses against antigen-expressing tumor targets (A) The percentages of CD107a-expressing, degranulating NK cells were determined by flow cytometry after 4-hour coincubation of nontransduced (NT), CD19-ζ, CD19-2B4, CD19-t2B4, CD19-2B4ζ, and CD19-t2B4ζ transduced NK cells with CD19-negative ML-2 leukemia cells, CD19+ REH leukemia cells, or K562 cells at an effector-to-target (E:T) ratio of 1:1, or in the presence of medium alone, as indicated. To exclude non-NK lymphocytes and nontransduced NK cells within the cultures from analysis, the gate was set on GFP/CD56-coexpressing cells. Shown is one representative experiment of two. (B) To directly assess the cytotoxic effects of transduced NK cells on CD19+ leukemia cells, FACS-purified (GFP+) NK cells expressing the various CARs were coincubated for 16 hours with the CD19+ leukemia cell line REH at a 1:3 E:T ratio. Each data point represents the mean percentage of target cytolysis compared to parallel cultures of REH cells in the absence of NK cells. Non-transduced NK cells were used as negative controls. Shown is one representative experiment of two, each performed in duplicate wells.
    Figure Legend Snippet: CAR transduced NK cells show powerful cytolytic responses against antigen-expressing tumor targets (A) The percentages of CD107a-expressing, degranulating NK cells were determined by flow cytometry after 4-hour coincubation of nontransduced (NT), CD19-ζ, CD19-2B4, CD19-t2B4, CD19-2B4ζ, and CD19-t2B4ζ transduced NK cells with CD19-negative ML-2 leukemia cells, CD19+ REH leukemia cells, or K562 cells at an effector-to-target (E:T) ratio of 1:1, or in the presence of medium alone, as indicated. To exclude non-NK lymphocytes and nontransduced NK cells within the cultures from analysis, the gate was set on GFP/CD56-coexpressing cells. Shown is one representative experiment of two. (B) To directly assess the cytotoxic effects of transduced NK cells on CD19+ leukemia cells, FACS-purified (GFP+) NK cells expressing the various CARs were coincubated for 16 hours with the CD19+ leukemia cell line REH at a 1:3 E:T ratio. Each data point represents the mean percentage of target cytolysis compared to parallel cultures of REH cells in the absence of NK cells. Non-transduced NK cells were used as negative controls. Shown is one representative experiment of two, each performed in duplicate wells.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, FACS, Purification

    Expansion and immunophenotypes of NK cells cocultured with K562-mb15-41BBL cells (A) Peripheral blood mononuclear cells from five healthy donors and from four pediatric patients in first hematological remission of B-cell precursor ALL were cocultured with irradiated K562mb15-4-1BBL at a 0.75:1 ratio in the presence of rhIL-2 (40 IU/ml), and absolute numbers of NK cells were calculated every 5 days after staining of viable cells with CD3- and CD56-specific antibodies. (B) Cell surface expression of CD3 and CD56 prior to and on days 5 and 10 after stimulation by flow cytometry, and expression of CD56, CD16, CD244 (2B4) and CD48 on day 10 of coculture with K562-mb15-41BBL cells. Shown are representative histograms for one of five cell cultures obtained from four donors.
    Figure Legend Snippet: Expansion and immunophenotypes of NK cells cocultured with K562-mb15-41BBL cells (A) Peripheral blood mononuclear cells from five healthy donors and from four pediatric patients in first hematological remission of B-cell precursor ALL were cocultured with irradiated K562mb15-4-1BBL at a 0.75:1 ratio in the presence of rhIL-2 (40 IU/ml), and absolute numbers of NK cells were calculated every 5 days after staining of viable cells with CD3- and CD56-specific antibodies. (B) Cell surface expression of CD3 and CD56 prior to and on days 5 and 10 after stimulation by flow cytometry, and expression of CD56, CD16, CD244 (2B4) and CD48 on day 10 of coculture with K562-mb15-41BBL cells. Shown are representative histograms for one of five cell cultures obtained from four donors.

    Techniques Used: Irradiation, Staining, Expressing, Flow Cytometry, Cytometry

    11) Product Images from "Curcumin Induces Pro-apoptotic Effects Against Human Melanoma Cells and Modulates The Cellular Response to Immunotherapeutic Cytokines"

    Article Title: Curcumin Induces Pro-apoptotic Effects Against Human Melanoma Cells and Modulates The Cellular Response to Immunotherapeutic Cytokines

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-09-0377

    Curcumin inhibits functional properties of human NK cells (A) Levels of IFN-γ were assayed in the culture supernatants of NK cells from normal human donors following a 24 hour co-culture with curcumin (10 or 20 µM) or recombinant human IL-12 (10 or 50 ng/mL). Cells stimulated with DMSO and PBS (vehicles) served as negative controls. Error bars represent data combined data derived from two separate donors. Detection of (B) granzyme B (GrB) and (C) IFN-γ release from NK cells by ELISPOT. Following overnight culture with DMSO (vehicle) or curcumin (20 µM), NK cells from normal donors were cultured with A375 human melanoma cells or K562 cells as targets at a 20:1 effector:target ratio. Data shown are derived from independent experiments with NK cells from n = 3 normal donors.
    Figure Legend Snippet: Curcumin inhibits functional properties of human NK cells (A) Levels of IFN-γ were assayed in the culture supernatants of NK cells from normal human donors following a 24 hour co-culture with curcumin (10 or 20 µM) or recombinant human IL-12 (10 or 50 ng/mL). Cells stimulated with DMSO and PBS (vehicles) served as negative controls. Error bars represent data combined data derived from two separate donors. Detection of (B) granzyme B (GrB) and (C) IFN-γ release from NK cells by ELISPOT. Following overnight culture with DMSO (vehicle) or curcumin (20 µM), NK cells from normal donors were cultured with A375 human melanoma cells or K562 cells as targets at a 20:1 effector:target ratio. Data shown are derived from independent experiments with NK cells from n = 3 normal donors.

    Techniques Used: Functional Assay, Co-Culture Assay, Recombinant, Derivative Assay, Enzyme-linked Immunospot, Cell Culture

    12) Product Images from "Discovery of a New Drug-like Series of OGT Inhibitors by Virtual Screening"

    Article Title: Discovery of a New Drug-like Series of OGT Inhibitors by Virtual Screening

    Journal: Molecules

    doi: 10.3390/molecules27061996

    Cellular activity of OGT inhibitor 1. ( a ) Metabolic activity after treating K562 and AMO1 cells with 1 and OSMI-4b for 72 h at 40 and 20 µM. The results are presented as the percentage of metabolic activity of the control cells stimulated with the vehicle (mean + SD) from two independent experiments. ( b ) Representative picture of Western blot analysis of O -GlcNAc levels after treating AMO1 cells with 1 or vehicle for 4 h (two independent experiments). ß-tubulin was used as the loading control. ( c ) The Western blot results are presented as the relative level of O -GlcNAcylation of the control AMO1 cells stimulated with the vehicle for two independent experiments. OSMI-4b was used as the positive control ( Figure S7 ).
    Figure Legend Snippet: Cellular activity of OGT inhibitor 1. ( a ) Metabolic activity after treating K562 and AMO1 cells with 1 and OSMI-4b for 72 h at 40 and 20 µM. The results are presented as the percentage of metabolic activity of the control cells stimulated with the vehicle (mean + SD) from two independent experiments. ( b ) Representative picture of Western blot analysis of O -GlcNAc levels after treating AMO1 cells with 1 or vehicle for 4 h (two independent experiments). ß-tubulin was used as the loading control. ( c ) The Western blot results are presented as the relative level of O -GlcNAcylation of the control AMO1 cells stimulated with the vehicle for two independent experiments. OSMI-4b was used as the positive control ( Figure S7 ).

    Techniques Used: Activity Assay, Western Blot, Positive Control

    13) Product Images from "A c-Myb mutant causes deregulated differentiation due to impaired histone binding and abrogated pioneer factor function"

    Article Title: A c-Myb mutant causes deregulated differentiation due to impaired histone binding and abrogated pioneer factor function

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx364

    Comparison of the transcriptomes associated with c-Myb WT and D152V generated by RNA-seq following c-Myb knockdown and rescue in K562 cells. ( A ) Expression of MYB analysed by qRT-PCR and western blotting using anti-c-Myb and anti-GAPDH primary antibodies. RNA and cell lysates were harvested 24 h after transfection with siRNA. RNA was isolated from three independent biological replicates, delivered to high-throughput sequencing and subjected to downstream statistical processing, as described in ‘Materials and Methods’ section. ( B ) Number of genes differentially expressed after knockdown of c-Myb rescued by ectopic expression of TY-c-Myb and TY-c-Myb D152V (766), as well as the number of genes not rescued by TY-c-Myb D152V (104). ( C ) Heat map of the differential gene expression pattern after c-Myb knockdown of the genes rescued by TY-c-Myb, but not by TY-c-Myb-D152V. Yellow colour represents decreased expression while blue colour represents increased expression relative to the control (black). ( D ) RNA-seq data showing the expression pattern of genes differentially expressed after c-Myb knockdown rescued by expression of both TY-c-Myb and TY-c-Myb D152V. The representative genes shown are MYB, OGDH, PNPT1, RELA and H2AFZ . To illustrate the rescue for individual genes, we extracted data for each replicate to estimate mean ± SD. Significance was evaluated by unpaired, two-tailed t -tests on selected pairs and indicated with P -values (* P
    Figure Legend Snippet: Comparison of the transcriptomes associated with c-Myb WT and D152V generated by RNA-seq following c-Myb knockdown and rescue in K562 cells. ( A ) Expression of MYB analysed by qRT-PCR and western blotting using anti-c-Myb and anti-GAPDH primary antibodies. RNA and cell lysates were harvested 24 h after transfection with siRNA. RNA was isolated from three independent biological replicates, delivered to high-throughput sequencing and subjected to downstream statistical processing, as described in ‘Materials and Methods’ section. ( B ) Number of genes differentially expressed after knockdown of c-Myb rescued by ectopic expression of TY-c-Myb and TY-c-Myb D152V (766), as well as the number of genes not rescued by TY-c-Myb D152V (104). ( C ) Heat map of the differential gene expression pattern after c-Myb knockdown of the genes rescued by TY-c-Myb, but not by TY-c-Myb-D152V. Yellow colour represents decreased expression while blue colour represents increased expression relative to the control (black). ( D ) RNA-seq data showing the expression pattern of genes differentially expressed after c-Myb knockdown rescued by expression of both TY-c-Myb and TY-c-Myb D152V. The representative genes shown are MYB, OGDH, PNPT1, RELA and H2AFZ . To illustrate the rescue for individual genes, we extracted data for each replicate to estimate mean ± SD. Significance was evaluated by unpaired, two-tailed t -tests on selected pairs and indicated with P -values (* P

    Techniques Used: Generated, RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Isolation, Next-Generation Sequencing, Two Tailed Test

    Hemin-induced erythroid differentiation of K562 cells. ( A ) K562 cells were induced for erythroid differentiation with 30 μM hemin for 72 h. The erythroid differentiation is evident by the red cell pellets. ( B ) Seventy-two hours after induction with hemin, the cells were stained with benzidine and the haemoglobin-positive cells (black, some with spikes) were measured. The number of benzidine-positive cells was estimated by manually counting triplicates of 300 cells in a light microscope. Three biological replicates were analysed (total of 900 cells). ( C ) Expression of MYB analysed by qRT-PCR and protein level of c-Myb analysed by western blotting using anti-c-Myb and anti-GAPDH primary antibodies 72 h after induction with hemin in the control K562 cell line. ( D ) The amount of haemoglobin-positive cells, counted as described above, presented as percentage of differentiated cells. Cell pellets are also shown to visualize the degree of differentiation. All cell counting and qRT-PCR results are presented as mean ± SD of three independent biological replicates. P -values are indicated as in Figure 1D .
    Figure Legend Snippet: Hemin-induced erythroid differentiation of K562 cells. ( A ) K562 cells were induced for erythroid differentiation with 30 μM hemin for 72 h. The erythroid differentiation is evident by the red cell pellets. ( B ) Seventy-two hours after induction with hemin, the cells were stained with benzidine and the haemoglobin-positive cells (black, some with spikes) were measured. The number of benzidine-positive cells was estimated by manually counting triplicates of 300 cells in a light microscope. Three biological replicates were analysed (total of 900 cells). ( C ) Expression of MYB analysed by qRT-PCR and protein level of c-Myb analysed by western blotting using anti-c-Myb and anti-GAPDH primary antibodies 72 h after induction with hemin in the control K562 cell line. ( D ) The amount of haemoglobin-positive cells, counted as described above, presented as percentage of differentiated cells. Cell pellets are also shown to visualize the degree of differentiation. All cell counting and qRT-PCR results are presented as mean ± SD of three independent biological replicates. P -values are indicated as in Figure 1D .

    Techniques Used: Staining, Light Microscopy, Expressing, Quantitative RT-PCR, Western Blot, Cell Counting

    Differential chromatin accessibility revealed by ATAC-seq ( A ) Regions with ≥50% fold difference after knockdown of c-Myb (comparing K562 cells transfected with control siRNA and si2992) showing decreased chromatin accessibility (910) or increased chromatin accessibility ( 54 ). ( B ) Regions with ≥50% fold difference between WT c-Myb rescue and the D152V rescue (comparing the K562 cell lines ectopically expressing TY-tagged versions of c-Myb after knockdown of endogenous c-Myb) showing decreased chromatin accessibility (2295) in D152V compared to WT or increased chromatin accessibility (491) in D152V compared to WT. ( C ) Venn diagram showing the overlap (692) between the 910 regions with decreased chromatin accessibility upon c-Myb knockdown and the 2295 regions with decreased chromatin accessibility upon D152V rescue compared to WT c-Myb rescue. ( D ) Genomic distribution (defined by diffReps) of the 692 overlapping regions. ( E ) The frequency of the c-Myb binding motif shown, for the indicated subgroup ( n = 692). Motif analysis around peak regions for the intersection of differential ATAC peak regions between MYB knockdown and D152V was performed using the HOMER program ( 3 ), as described in ‘Materials and Methods’ section. Values for the n = 1603 and n = 910 groups were 71 and 64% respectively. ( F ) UCSC Genome Browser tracks of the AMER1, GLIS2 and LMO2 loci showing ATAC-seq signal of K562 cells transfected with control siRNA, si2992 (MYB siRNA), as well as the WT c-Myb and D152V rescues. DNase I hypersensitivity (HS) mapping from K562 cells and ChIP-seq data on H3K27Ac in K562 cells (generated by the ENCODE project) is also shown.
    Figure Legend Snippet: Differential chromatin accessibility revealed by ATAC-seq ( A ) Regions with ≥50% fold difference after knockdown of c-Myb (comparing K562 cells transfected with control siRNA and si2992) showing decreased chromatin accessibility (910) or increased chromatin accessibility ( 54 ). ( B ) Regions with ≥50% fold difference between WT c-Myb rescue and the D152V rescue (comparing the K562 cell lines ectopically expressing TY-tagged versions of c-Myb after knockdown of endogenous c-Myb) showing decreased chromatin accessibility (2295) in D152V compared to WT or increased chromatin accessibility (491) in D152V compared to WT. ( C ) Venn diagram showing the overlap (692) between the 910 regions with decreased chromatin accessibility upon c-Myb knockdown and the 2295 regions with decreased chromatin accessibility upon D152V rescue compared to WT c-Myb rescue. ( D ) Genomic distribution (defined by diffReps) of the 692 overlapping regions. ( E ) The frequency of the c-Myb binding motif shown, for the indicated subgroup ( n = 692). Motif analysis around peak regions for the intersection of differential ATAC peak regions between MYB knockdown and D152V was performed using the HOMER program ( 3 ), as described in ‘Materials and Methods’ section. Values for the n = 1603 and n = 910 groups were 71 and 64% respectively. ( F ) UCSC Genome Browser tracks of the AMER1, GLIS2 and LMO2 loci showing ATAC-seq signal of K562 cells transfected with control siRNA, si2992 (MYB siRNA), as well as the WT c-Myb and D152V rescues. DNase I hypersensitivity (HS) mapping from K562 cells and ChIP-seq data on H3K27Ac in K562 cells (generated by the ENCODE project) is also shown.

    Techniques Used: Transfection, Expressing, Binding Assay, Chromatin Immunoprecipitation, Generated

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    ATCC k562
    Evaluation of the genome-editing activity of nucleofected multi-NLS SpCas9 proteins. ( A , B ) Evaluation of the genome-editing activity of multi-NLS SpCas9 at the CCR5 site in <t>K562</t> ( A ) and Jurkat ( B ) cells. The results are quantified using T7E1 analysis and presented as mean ± SD ( n = 2). The difference between N0/C1 and multi-NLS constructs is analyzed by two-tailed Student’s t test. *, p
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    Evaluation of the genome-editing activity of nucleofected multi-NLS SpCas9 proteins. ( A , B ) Evaluation of the genome-editing activity of multi-NLS SpCas9 at the CCR5 site in K562 ( A ) and Jurkat ( B ) cells. The results are quantified using T7E1 analysis and presented as mean ± SD ( n = 2). The difference between N0/C1 and multi-NLS constructs is analyzed by two-tailed Student’s t test. *, p

    Journal: Bioengineering

    Article Title: Systematic Investigation of the Effects of Multiple SV40 Nuclear Localization Signal Fusion on the Genome Editing Activity of Purified SpCas9

    doi: 10.3390/bioengineering9020083

    Figure Lengend Snippet: Evaluation of the genome-editing activity of nucleofected multi-NLS SpCas9 proteins. ( A , B ) Evaluation of the genome-editing activity of multi-NLS SpCas9 at the CCR5 site in K562 ( A ) and Jurkat ( B ) cells. The results are quantified using T7E1 analysis and presented as mean ± SD ( n = 2). The difference between N0/C1 and multi-NLS constructs is analyzed by two-tailed Student’s t test. *, p

    Article Snippet: K562 and Jurkat cells were acquired from American Type Culture Collection (ATCC) and maintained in RPMI 1640 medium containing 10% (v /v ) fetal bovine serum (FBS, Gibco), 100 U/mL penicillin and 100 U/mL streptomycin.

    Techniques: Activity Assay, Construct, Two Tailed Test

    Evaluation of the effects of multi-NLS on SpCas9 activity and specificity in K562 cells. ( A ) The gene-editing activities of multi-NLS on SpCas9 at different dosage, as determined by T7E1 analysis. The data are shown as mean ± SD ( n = 3 technical replicates). ( B ) The effects of multi-NLS on the specificity of SpCas9 variants. The specificity is determined by the ratio between on-target and off-target activities. The difference between N0/C1 and multi-NLS constructs at each corresponding condition is analyzed by one-way ANOVA with Bonferroni’s multiple comparisons test. *, p

    Journal: Bioengineering

    Article Title: Systematic Investigation of the Effects of Multiple SV40 Nuclear Localization Signal Fusion on the Genome Editing Activity of Purified SpCas9

    doi: 10.3390/bioengineering9020083

    Figure Lengend Snippet: Evaluation of the effects of multi-NLS on SpCas9 activity and specificity in K562 cells. ( A ) The gene-editing activities of multi-NLS on SpCas9 at different dosage, as determined by T7E1 analysis. The data are shown as mean ± SD ( n = 3 technical replicates). ( B ) The effects of multi-NLS on the specificity of SpCas9 variants. The specificity is determined by the ratio between on-target and off-target activities. The difference between N0/C1 and multi-NLS constructs at each corresponding condition is analyzed by one-way ANOVA with Bonferroni’s multiple comparisons test. *, p

    Article Snippet: K562 and Jurkat cells were acquired from American Type Culture Collection (ATCC) and maintained in RPMI 1640 medium containing 10% (v /v ) fetal bovine serum (FBS, Gibco), 100 U/mL penicillin and 100 U/mL streptomycin.

    Techniques: Activity Assay, Construct

    HMG2 is cleaved in cells perforin loaded with GzmA. (a) K562 cells were treated for the indicated times at 37°C with 1 μM GzmA (A) or 1 μM S-AGzmA (S-A) and sublytic concentrations of perforin (P). Cell lysates were analyzed by immunoblotting for HMG2, SET, and pp32. Neither full-length HMG2 nor SET are detected after 2 h, but pp32 is unchanged. (b) Nuclear fractions were isolated 4 h after GzmA loading with perforin and analyzed by immunoblotting. Nuclear SET and HMG2 are completely degraded by active, but not inactive, enzyme in a perforin-dependent manner.

    Journal: Molecular and Cellular Biology

    Article Title: HMG2 Interacts with the Nucleosome Assembly Protein SET and Is a Target of the Cytotoxic T-Lymphocyte Protease Granzyme A

    doi: 10.1128/MCB.22.8.2810-2820.2002

    Figure Lengend Snippet: HMG2 is cleaved in cells perforin loaded with GzmA. (a) K562 cells were treated for the indicated times at 37°C with 1 μM GzmA (A) or 1 μM S-AGzmA (S-A) and sublytic concentrations of perforin (P). Cell lysates were analyzed by immunoblotting for HMG2, SET, and pp32. Neither full-length HMG2 nor SET are detected after 2 h, but pp32 is unchanged. (b) Nuclear fractions were isolated 4 h after GzmA loading with perforin and analyzed by immunoblotting. Nuclear SET and HMG2 are completely degraded by active, but not inactive, enzyme in a perforin-dependent manner.

    Article Snippet: K562 and HeLa cells were obtained from American Type Culture Collection and grown in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine, 2 mM HEPES, 100 U of penicillin per ml, 100 mg of streptomycin per ml, and 50 μM β-mercaptoethanol.

    Techniques: Isolation

    GzmA, but not GzmB, cleaves native HMG2 in K562 cell lysates and isolated nuclei. (a) K562 postnuclear cytoplasmic lysates (2 × 10 5 equivalents) were incubated with the indicated concentrations of GzmA or 1 μM S-AGzmA or GzmB at 37°C for 2 h and analyzed by immunoblotting for HMG2, SET, and pp32. HMG2 and SET are cleaved at nanomolar concentrations of GzmA, but pp32 is unchanged. (b) Increasing concentrations of GzmA or 1 μM inactive S-AGzmA was incubated with 10 6 K562 nuclei and nuclear lysates were analyzed 1.5 h later for cleavage of HMG2, SET, pp32, and HMG1. Full-length SET and HMG2 are completely cleaved at nanomolar concentrations of GzmA.

    Journal: Molecular and Cellular Biology

    Article Title: HMG2 Interacts with the Nucleosome Assembly Protein SET and Is a Target of the Cytotoxic T-Lymphocyte Protease Granzyme A

    doi: 10.1128/MCB.22.8.2810-2820.2002

    Figure Lengend Snippet: GzmA, but not GzmB, cleaves native HMG2 in K562 cell lysates and isolated nuclei. (a) K562 postnuclear cytoplasmic lysates (2 × 10 5 equivalents) were incubated with the indicated concentrations of GzmA or 1 μM S-AGzmA or GzmB at 37°C for 2 h and analyzed by immunoblotting for HMG2, SET, and pp32. HMG2 and SET are cleaved at nanomolar concentrations of GzmA, but pp32 is unchanged. (b) Increasing concentrations of GzmA or 1 μM inactive S-AGzmA was incubated with 10 6 K562 nuclei and nuclear lysates were analyzed 1.5 h later for cleavage of HMG2, SET, pp32, and HMG1. Full-length SET and HMG2 are completely cleaved at nanomolar concentrations of GzmA.

    Article Snippet: K562 and HeLa cells were obtained from American Type Culture Collection and grown in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine, 2 mM HEPES, 100 U of penicillin per ml, 100 mg of streptomycin per ml, and 50 μM β-mercaptoethanol.

    Techniques: Isolation, Incubation

    ). (b) rHMG2 binds directly to rSET. rHMG2 and rSET were coincubated and immunoprecipitated with anti-HMG2 antisera (left) or anti-SET monoclonal antibody (right) or control antibody. (c) The experiment shown in panel b was repeated with K562 postnuclear lysates to coprecipitate native SET and HMG2. (d) However, despite the close homology between HMG1 and HMG2, HMG1 and SET do not coprecipitate from K562 cell lysates. (e) rHMG2 does not bind directly to rAPE, another SET complex protein (Fan et al., submitted).

    Journal: Molecular and Cellular Biology

    Article Title: HMG2 Interacts with the Nucleosome Assembly Protein SET and Is a Target of the Cytotoxic T-Lymphocyte Protease Granzyme A

    doi: 10.1128/MCB.22.8.2810-2820.2002

    Figure Lengend Snippet: ). (b) rHMG2 binds directly to rSET. rHMG2 and rSET were coincubated and immunoprecipitated with anti-HMG2 antisera (left) or anti-SET monoclonal antibody (right) or control antibody. (c) The experiment shown in panel b was repeated with K562 postnuclear lysates to coprecipitate native SET and HMG2. (d) However, despite the close homology between HMG1 and HMG2, HMG1 and SET do not coprecipitate from K562 cell lysates. (e) rHMG2 does not bind directly to rAPE, another SET complex protein (Fan et al., submitted).

    Article Snippet: K562 and HeLa cells were obtained from American Type Culture Collection and grown in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine, 2 mM HEPES, 100 U of penicillin per ml, 100 mg of streptomycin per ml, and 50 μM β-mercaptoethanol.

    Techniques: Immunoprecipitation

    HMG2 coelutes with the SET complex proteins SET and pp32. (a) A 270- to 420-kDa complex elutes from K562 cell lysates applied sequentially to immobilized S-AGzmA and S400 gel filtration columns. Migration of Pharmacia gel filtration standards is indicated. (b) The SET complex, isolated from the S-AGzmA affinity column, is disrupted by purification on an anion-exchange column. The acidic SET and pp32 proteins stick to the anion-exchange column (eluate, E), but most proteins are in the flowthrough (FT). HMG2 was identified by N-terminal sequencing of a prominent 28-kDa band in the flowthrough. The identification of the indicated bands was verified by immunoblotting (not shown). (c) Immunoblots confirm the comigration of HMG2, SET, and pp32 in the S400 column fractions.

    Journal: Molecular and Cellular Biology

    Article Title: HMG2 Interacts with the Nucleosome Assembly Protein SET and Is a Target of the Cytotoxic T-Lymphocyte Protease Granzyme A

    doi: 10.1128/MCB.22.8.2810-2820.2002

    Figure Lengend Snippet: HMG2 coelutes with the SET complex proteins SET and pp32. (a) A 270- to 420-kDa complex elutes from K562 cell lysates applied sequentially to immobilized S-AGzmA and S400 gel filtration columns. Migration of Pharmacia gel filtration standards is indicated. (b) The SET complex, isolated from the S-AGzmA affinity column, is disrupted by purification on an anion-exchange column. The acidic SET and pp32 proteins stick to the anion-exchange column (eluate, E), but most proteins are in the flowthrough (FT). HMG2 was identified by N-terminal sequencing of a prominent 28-kDa band in the flowthrough. The identification of the indicated bands was verified by immunoblotting (not shown). (c) Immunoblots confirm the comigration of HMG2, SET, and pp32 in the S400 column fractions.

    Article Snippet: K562 and HeLa cells were obtained from American Type Culture Collection and grown in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine, 2 mM HEPES, 100 U of penicillin per ml, 100 mg of streptomycin per ml, and 50 μM β-mercaptoethanol.

    Techniques: Filtration, Migration, Isolation, Affinity Column, Purification, Sequencing, Western Blot

    sCAR19 T cells exhibit cytotoxicity in vitro . (a) Cytotoxicity of un-transduced T cells, CAR19 T cells and sCAR19 T cells cultured in two conditions, one with the DMSO vehicle and the other with 1 μM ASV toward CD19 + Raji (the left graph) and CD19 - K562 cells (the right graph). Both Raji and K562 cells were labeled with calcein-AM before they were cocultured with four groups of T cells with ratios of effector to target tumor cells (E:T) as indicated in the figures for 4 h. Lysis of target cells was analyzed by detecting released calcein-AM in media. Data are representative of three independent experiments and normalized against total lysis of calcein-AM-labeled Raji and K562 cells. (b) The release of cytokines including IFN-γ, IL-2 and TNFα from un-transduced T cells, CAR19 T cells and sCAR19 T cells cultured in two conditions, one with the DMSO vehicle and the other with 1 μM ASV when they were cocultured with Raji cells with a E:T ratio as 1:1 for 24 h. Cytokine levels were detected using ELISA.

    Journal: bioRxiv

    Article Title: A Recurring Chemogenetic Switch for Chimeric Antigen Receptor T Cells

    doi: 10.1101/2021.08.23.457355

    Figure Lengend Snippet: sCAR19 T cells exhibit cytotoxicity in vitro . (a) Cytotoxicity of un-transduced T cells, CAR19 T cells and sCAR19 T cells cultured in two conditions, one with the DMSO vehicle and the other with 1 μM ASV toward CD19 + Raji (the left graph) and CD19 - K562 cells (the right graph). Both Raji and K562 cells were labeled with calcein-AM before they were cocultured with four groups of T cells with ratios of effector to target tumor cells (E:T) as indicated in the figures for 4 h. Lysis of target cells was analyzed by detecting released calcein-AM in media. Data are representative of three independent experiments and normalized against total lysis of calcein-AM-labeled Raji and K562 cells. (b) The release of cytokines including IFN-γ, IL-2 and TNFα from un-transduced T cells, CAR19 T cells and sCAR19 T cells cultured in two conditions, one with the DMSO vehicle and the other with 1 μM ASV when they were cocultured with Raji cells with a E:T ratio as 1:1 for 24 h. Cytokine levels were detected using ELISA.

    Article Snippet: Cell line culture We purchased K562, Raji and HEK 293T/17 from American Type Culture Collection.

    Techniques: In Vitro, Cell Culture, Labeling, Lysis, Enzyme-linked Immunosorbent Assay

    Reconstruction of a complex Philadelphia chromosome. a AA-generated breakpoint graph for K562. Estimated copy number (CN), coverage, discordant reads forming breakpoint graph edges, and a subset of the genes in these regions are shown. b AR reconstruction of an 8.5 Mbp focal amplification which was supported by both Irys and Saphyr reconstructions. The tracks from top to bottom are: OM contigs (with contig ID and direction indicated above), graph segments (alignments shown with vertical gray lines), gene subset, and color-coded reference genome bar with genomic coordinates (scaled as 10 kbp units). Gray half-height bars between individual segments on the reference genome bar indicate support from edges in the AA breakpoint graph. White arrows inside the chromosome color bar indicate direction of genomic segment(s). Colored numbers correspond to numbered breakpoint graph edges in panel ( a ). c Multi-FISH using probes against BCR , ABL1 , and GPC5 with DAPI-stained metaphase chromosomes. Scale bars indicate 2 µm in both “Full size” and “Zoom” rows.

    Journal: Nature Communications

    Article Title: AmpliconReconstructor integrates NGS and optical mapping to resolve the complex structures of focal amplifications

    doi: 10.1038/s41467-020-18099-z

    Figure Lengend Snippet: Reconstruction of a complex Philadelphia chromosome. a AA-generated breakpoint graph for K562. Estimated copy number (CN), coverage, discordant reads forming breakpoint graph edges, and a subset of the genes in these regions are shown. b AR reconstruction of an 8.5 Mbp focal amplification which was supported by both Irys and Saphyr reconstructions. The tracks from top to bottom are: OM contigs (with contig ID and direction indicated above), graph segments (alignments shown with vertical gray lines), gene subset, and color-coded reference genome bar with genomic coordinates (scaled as 10 kbp units). Gray half-height bars between individual segments on the reference genome bar indicate support from edges in the AA breakpoint graph. White arrows inside the chromosome color bar indicate direction of genomic segment(s). Colored numbers correspond to numbered breakpoint graph edges in panel ( a ). c Multi-FISH using probes against BCR , ABL1 , and GPC5 with DAPI-stained metaphase chromosomes. Scale bars indicate 2 µm in both “Full size” and “Zoom” rows.

    Article Snippet: Cell cultureNCI-H460, K562, and HCC827 cells were obtained from ATCC and cultured in RPMI-1640 media supplemented with 10% FBS.

    Techniques: Generated, Amplification, Fluorescence In Situ Hybridization, Staining