myelogenous leukemia cell line k562 cells  (ATCC)

Journal: Frontiers in Immunology

Article Title: Chemical priming potentiates mesothelin-targeting chimeric antigen receptor-engineered NK-92 antitumor activity by improving tumor trafficking and cytotoxic killing dynamics

doi: 10.3389/fimmu.2026.1860442

Figure Lengend Snippet: 25KbPEI-mediated chemical priming enhances the cytotoxicity of MSLN-targeting CAR-NK-92. (A) Cytotoxic activity of parental NK-92 and CAR-NK-92 against K562 cells was assessed using a Far Red/7-AAD assay (E:T ratio = 2:1). (B) Antigen-specific cytotoxicity of NK-92 and CAR-NK-92 against MSLN-positive ovarian cancer cell lines (OVCAR3 and SKOV3) was evaluated using a Far Red/7-AAD assay (E:T ratio = 10:1). (C) Schematic illustration of the generation of CAR-Chem_NK-92 by treatment of CAR-NK-92 cells with 25KbPEI. (D) Cell viability of CAR-NK-92 following 25KbPEI treatment was determined at 12 h post-treatment using a trypan blue exclusion assay. (E) Dose-dependent effects of 25KbPEI on CAR-NK-92 cytotoxicity were evaluated against OVCAR3 cells using a Far Red/7-AAD assay (E:T ratio = 10:1). (F) Comparative cytotoxic activity of NK-92, CAR-NK-92, and CAR-Chem_NK-92 against OVCAR3 and SKOV3 cells (E:T ratio = 10:1). (G) Degranulation activity of NK-92, CAR-NK-92, and CAR-Chem_NK-92 following co-culture with OVCAR3 and SKOV3 cells was assessed by CD107a surface expression (E:T ratio = 1:1). All experiments were performed in three independent biological replicates. Statistical significance was determined using an unpaired Student ’ s t-test for panels A–B and one-way ANOVA followed by Tukey ’ s multiple comparisons test for panels E–G. Data are presented as mean ± SD. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The human myelogenous leukemia cell line K562 and the human ovarian cancer cell lines OVCAR3 and SKOV3 were obtained from the American Type Culture Collection (ATCC).

Techniques: Activity Assay, Trypan Blue Exclusion Assay, Co-Culture Assay, Expressing

Journal: bioRxiv

Article Title: TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma

doi: 10.64898/2026.05.29.728710

Figure Lengend Snippet: Results from K562/luc (expressing shNEG, Firefly luciferase, and GFP) alone (mono) or mixed with human primary bone marrow mesenchymal stem cells (with hMSC) were subcutaneously injected in mice to form xenografts. (A) Scheme demonstrating the experimental setup; samples were collected 21 days after subcutaneous injection of cells in mice. (B) Weight of xenografts formed by K562/luc mono or a mix with hMSCs in mice (n=5 in each variant), and the two-tailed, nonparametric Mann-Whitney test was used to check the difference significance; the exact significance ( P ) values are presented in the plot. (C-H) Short-read sequencing RNA analysis results of samples from cells isolated from xenografts mono and with hMSC treated for the last 14 days with vehicle (shNEG) by the fluorescence-activated cell sorting (FACS) with BD Aria. (C) Representative scatterplots (n=3) with the gating strategy used for the FACS-sorting of live xenograft cells (negative for Viability dye-eFluor780 staining), expressing GFP (GFP+) and stained positive with the antibody against human CD45 with BV421 fluorophore (hCD45+). (D-G) Results of differential expression analysis of the variant shNEG versus mono by DeSeq2 of RNA sequenced from FACS-sorted cells. Presented are the results from cells isolated from three different xenografts for each variant. (D) Principal component analysis. (E) Number of genes with fold change absolute value ≥ 50% and p-value corrected for multiple testing using the Benjamini-Hochberg P adj. ≤ 0.05, that are upregulated (UP) or downregulated (DOWN) in cells from xenograft (Xgraft) shNEG versus mono. (F) Expression level (counts corrected for the sample sequencing depth) in each xenograft (n=3) of the top 150 genes (each row) with the most significant (P adj. ) fold change in expression level. (G) The top of Gene Ontology Biological Processes (GOBP) terms from the Gene Set Enrichment Analysis (GSEA) of the DeSeq2 results, with the highest positive (left) and negative (right) normalized enrichment score (NES), P adj. ≤ 0.05 (FDR) and number of genes in the sample annotated per term (size) > 20. The running enrichment score is presented by a grey line for each of the ranked genes marked with a vertical black line. (H) The Reactome terms identified by the GSEA of the UP or DOWN genes in the variant shNEG versus mono, with NES ≥ 1.5 (absolute value), p value ≤ 0.05, and size > 30.

Article Snippet: Human chronic myeloid leukemia cell line K562 (#CCL-243) and bone marrow stroma fibroblast cell line HS-5 (#CRL-11882) were obtained from American Type Culture Collection (USA); human chronic myeloid leukemia cell line LAMA-84 (#ACC 168) was from DSMZ.

Techniques: Expressing, Luciferase, Injection, Variant Assay, Two Tailed Test, MANN-WHITNEY, Sequencing, Isolation, Fluorescence, FACS, Staining, Quantitative Proteomics

Journal: bioRxiv

Article Title: TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma

doi: 10.64898/2026.05.29.728710

Figure Lengend Snippet: (A) Scheme explaining experimental steps of xenograft formation by K562/luc (expressing shNEG, Firefly luciferase, and GFP) mixed with human primary bone marrow mesenchymal stem cells (hMSC) subcutaneously injected in mice 7 days before initiation of treatment with IM for the following 14 days, when the GFP and human CD45 positive cells were FACS-sorted from xenografts for RNA isolation and sequencing. (B) Number of intersecting genes (compared groups indicated by black dot) that at the RNA level are upregulated (UP; log2 value of fold change ≥ 0.6) or downregulated (DOWN; log2 value of fold change ≤ -0.6) in shNEG cells xenografts imatinib versus vehicle treated and in CD34 positive cells from bone marrow biopsies of a patient with CML (data deposited at GEO under accession number GSE310243; from ) obtained after 6 months of imatinib therapy (combined samples SRR36072325 and SRR36072321) versus obtained at the diagnosis (combined SRR36072320 and SRR36072324). (B-D) Yellow shadow marks a group of genes that are UP or DOWN upon imatinib treatment in shNEG and CML cells (shNEG&CML), selected for subsequent analysis in (C) and (D). (C) Reactome terms identified by the GSEA analysis of the UP or DOWN genes in shNEG&CML with normalized enrichment score ≥ 1.5 (absolute value), p value ≤ 0.05, and number of genes in the sample annotated per term (size) ≥ 15. (C-D) The Reactome leading-edge genes identified by GSEA, annotated to terms marked with a brown triangle in (C) were selected to compare their expression level in single-cell RNA-seq data in (D) from bone marrow biopsies of healthy donors and CML patients with different responses to imatinib therapy. (D) Analysis of data at the Single-cell atlas of diagnostic Chronic Myeloid Leukemia bone marrow (scdbm) for CD34+ cells subtype . Data and detailed description of patient classification available: http://scdbm.ddnetbio.com ; A – responded to IM within 12 months; B – IM treatment failed within 18 months; C – resistant to IM and other TKI-s.

Article Snippet: Human chronic myeloid leukemia cell line K562 (#CCL-243) and bone marrow stroma fibroblast cell line HS-5 (#CRL-11882) were obtained from American Type Culture Collection (USA); human chronic myeloid leukemia cell line LAMA-84 (#ACC 168) was from DSMZ.

Techniques: Expressing, Luciferase, Injection, Isolation, Sequencing, Biomarker Discovery, Single Cell, RNA Sequencing, Diagnostic Assay

Journal: bioRxiv

Article Title: TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma

doi: 10.64898/2026.05.29.728710

Figure Lengend Snippet: (A) Colony formation by K562 cells expressing shRNA non-targeting (shNEG) or targeting mRNA of TIAL1 (shTIAR), TIA1 (shTIA-1), or FMR1 (shFMRP) that were collected from ex vivo hypoxic (1.5% O2) co-culture with HS-5 bone marrow stromal fibroblasts and treated with 1 μM imatinib (IM) or 50nM Talazoparib (BMN) added in two doses following the experimental scheme (left panel). Number of colonies in each of the 3 technical replicates from 3-4 independent biological experiments presented as % change relative to the untreated cells (dashed black line) set as 100 %. Student’s two-tailed t-test was used to compare two samples marked by the black line; #### or **** - p<0.0001, ns - p > 0.05. (B) Scheme explaining the experimental setup based on the subcutaneous implantation in mice of 3D printed scaffolds (photo taken with a Samsung mobile phone camera) seeded with human cells differentiated into osteoblasts and K562/luc cells with shNEG, shTIAR or shTIA-1, followed by IM treatment for 14 days. (C) Bioluminescence signal monitored in mice after 2 weeks of IM or vehicle treatment (timeline explained in (B) ) following luciferin injection, collected in the Burker’s Xtreme In-Vivo chamber for 30 sec, and overlaid on the mouse X-ray image. Scale presents the signal intensity of the color coding. (D) Sum of the signal intensity (P) collected per second (s) and area (mm 2 ) for each mouse analyzed (single dot) is presented. (E) Scheme explaining experimental steps of xenograft formation by K562/luc (expressing shNEG or shTIAR, Firefly luciferase, and GFP) mixed with human primary bone marrow mesenchymal stem cells (hMSC) subcutaneously injected in mice 7 days before initiation of treatment with IM for the following 14 days. (F) Weight of each xenograft isolated from mice (single dot) formed by K562/luc cells with shNEG or shTIAR (as in (E) ) presented as fold change of the weight mean value of xenografts from mice treated with vehicle; mean value indicated with the black line. The nonparametric two-tailed Mann-Whitney test was used for comparisons indicated by black lines underneath the exact significance ( P ) values are presented in the plot. (A,D,F) Two-way Anova was used to compare shTIAR upon IM versus other variants; @ - P < 0.002.

Article Snippet: Human chronic myeloid leukemia cell line K562 (#CCL-243) and bone marrow stroma fibroblast cell line HS-5 (#CRL-11882) were obtained from American Type Culture Collection (USA); human chronic myeloid leukemia cell line LAMA-84 (#ACC 168) was from DSMZ.

Techniques: Expressing, shRNA, Ex Vivo, Co-Culture Assay, Two Tailed Test, Injection, In Vivo, Luciferase, Isolation, MANN-WHITNEY

Journal: bioRxiv

Article Title: TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma

doi: 10.64898/2026.05.29.728710

Figure Lengend Snippet: (A) Comparison of gene expression in K562/luc cells with shNEG or shTIAR, FACS-sorted from xenografts (Xgraft), with level upon 2 weeks of IM versus vehicle (C) treatment changed with log2 value of fold change (Log2FC) ≥ 0.6 (UP) or ≤ -0.6 (DOWN); reversed regulation in shTIAR cells in purple. (B) Gene Ontology Molecular Function terms identified by the GSEA of genes that upon IM vs C in Xgraft of shNEG are reversely regulated in shTIAR (shaded in purple). In grey circles - UP (Log2FC > 0.6) n = 1993, brown circles DOWN (Log2FC < -0.6) n = 1756. Selected terms with NESabs ≥ 1.45, p-value ≤ 0.05, and the number of genes in the sample annotated per term (size) ≥ 10; n -number of genes. (C) Proportion of alternative splicing events (AS) changed upon shTIAR versus shNEG in cells from xenografts treated with vehicle (C) or imatinib (IM) for 2 weeks. Significant events from rMATS analysis requiring at least 20 reads/event, absolute change in PSI (Percent Spliced In) > 0.1 with FDR ≤ 0.05; total number in 3 experiments (n) above the bar. (D) Comparison of intron retention (RI; left panel) and cassette exon (CE; right panel) AS with significant PSI changed in shTIAR versus shNEG in cells from Xgraft or CO treated with IM. (E) Change in expression level of genes in Xgraft IM versus vehicle-treated for a subset of genes with significant changes shTIAR vs shNEG in RI (left panel) or SE (right panel) in cells from Xgraft and CO treated with IM; Log2FC ≤ -0.6 in brown, ≥ 0.6 in grey. (F) Difference in PSI value of CE, RI, and mutually exclusive exons (MXE) AS in: moro – shTIAR versus shNEG xenograft cells from mice treated with IM (Xgraft_IM); yellow – CD34 + enriched cells from bone marrow biopsies of a CML patient collected at 6-month IM therapy versus diagnosis GSE310243 (see in ). Included only with PSI > 0.1 and with FDR ≤ 0.05. (G) Expression level of genes with changes in AS (selected in (E) and (F) ) analyzed at the scdbm for the CD34 + cells subtype. Patient classification in .

Article Snippet: Human chronic myeloid leukemia cell line K562 (#CCL-243) and bone marrow stroma fibroblast cell line HS-5 (#CRL-11882) were obtained from American Type Culture Collection (USA); human chronic myeloid leukemia cell line LAMA-84 (#ACC 168) was from DSMZ.

Techniques: Comparison, Gene Expression, Alternative Splicing, Expressing, Biomarker Discovery

Journal: bioRxiv

Article Title: TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma

doi: 10.64898/2026.05.29.728710

Figure Lengend Snippet: (A) Scheme explaining experimental setup to determine proteomic changes using the quantitative BONCAT (QuaNCAT). K562 cells expressing shNEG or shTIAR growing in co-culture with HS-5 cells under hypoxia (1.5% O2) for 1.5 days were treated with imatinib (IM) for 18h before the bioorthogonal noncanonical amino acid tagging (BONCAT) of nascent proteins synthesized in cells for 4h. (B) Upper panel - number of genes and nascent proteins showing increased (UP; log₂ fold change [Log₂FC] ≥ 0.6) or decreased (DOWN; Log₂FC ≤ −0.6) abundance in IM-treated shTIAR cells compared with shNEG cells, with a significance threshold of p ≤ 0.05. In total, 2186 nascent proteins were quantified in the BONCAT experiment. Lower panel - scatter plot showing intensity-based absolute quantification (iBAQ), used as an estimate of relative protein molar abundance, plotted against Log₂FC values for BONCAT-identified proteins in shTIAR versus shNEG cells. Purple dots indicate hits with significant changes at both the protein and mRNA levels. Dashed vertical lines mark the Log₂FC thresholds of −0.6 and 0.6. (C) Functional annotation Gene Ontology Molecular Function enrichment analysis of UP (brown) or DOWN (purple) proteins with ClueGO/CytoScape, displaying proteins annotated with the term; only one side enriched terms with FDR < 0.05. (D) Lower panel - number of proteins that upon shTIAR are UP and DOWN regulated, for which mRNA was detected in the RNA immunoprecipitated (IP) in TIAR protein complexes (RIP), enriched in samples obtained with anti-TIAR antibody versus the same isotype non-binding antibody (ISO) (n=3647). Upper panel - example Image of Western blotting analysis of IP, with a sample of cell lysate used for IP loaded for reference (input). (E) Changes in the mRNA level, determined by real-time PCR and quantified using the ddCT method, expressed as log2 of change between IM-treated versus untreated cells, detected in samples from whole cells (upper panel) and anti-TIAR RIP (lower panel). Mean of 3 independent experiments with ± range is presented. Student’s t-test two-way was used to compare the difference between IM to C; * p ≤ 0.05, ** p≤0.005. (F) Number of genes identified in anti-TIAR RIP that show significant changes in intron retention (RI) or cassette exon alternative splicing (CE) in shTIAR versus shNEG alternative splicing analysis of RNA from IM-treated cells. Number of genes in the intersection provided above the bar; comparisons indicated by the black dot. (G) Sashimi plot (middle panel) demonstrating splicing of EIF4A2 mRNA within the region encompassing exons 8-11 and 3’UTR in RNA from anti-TIAR RIP. Alternative splice site usage marked by the purple line, and the percent usage ± ME (n=3) in numbers by the lines, reads coverage from 0-145 shown in grey, alternatively spliced exon marked by shaded yellow. Gene region scheme in the top. (H) Percent transcripts with TIAR-dependent alternative exon inclusion in K562 cells from xenografts treated with IM. Student’s t-test was used to compare results from three experiments (each dot) for the mean value marked with a thick black line; * p = 0.021.

Article Snippet: Human chronic myeloid leukemia cell line K562 (#CCL-243) and bone marrow stroma fibroblast cell line HS-5 (#CRL-11882) were obtained from American Type Culture Collection (USA); human chronic myeloid leukemia cell line LAMA-84 (#ACC 168) was from DSMZ.

Techniques: Expressing, Co-Culture Assay, Synthesized, Quantitative Proteomics, Functional Assay, Immunoprecipitation, Binding Assay, Western Blot, Real-time Polymerase Chain Reaction, Alternative Splicing

Journal: bioRxiv

Article Title: TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma

doi: 10.64898/2026.05.29.728710

Figure Lengend Snippet: (A) Expression level of genes analyzed at the scdbm for CD34 + cells subtype; patient classification explained in . (B-H) Impact of shTIAR compared to shNEG analyzed in K562 cells that were co-cultured with HS-5 cells under hypoxia (1.5%O2) 2 days before initiation of 18h culture with imatinib (IM) treatment or without (C). (B) Protein level in whole cell extracts analyzed by Western blot, representative images of immunoblots (n=3) presented. (C) Mitochondrial (mit.) membrane potential measured using JC-1 probe; values for signal from probe aggregates (red) in polarized mitochondria expressed as % of signal from the probe in the cells (n=3), and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) used as a control. (C,D,F,H) Bars represent the mean of values from independent experiments (indicated by dots) with ± SD. Student’s t-test was used to determine the significance of the difference shTIAR vs shNEG (#) and IM vs C (*); * - p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.005, **** p ≤ 0.001 (D) Lipid peroxidation measured with click-it chemistry by flow cytometry. Fluorescence intensity GeoMean expressed as fold change of value in the untreated shNEG cells (n=5). (E-H) Presentation on the cluster of cell differentiation (CD) surface protein markers CD45 and CD235a on K562 cells with shNEG or shTIAR isolated from co-culture established in hypoxia (1.5%O2) a day before initiation of treatment with IM for 48h (E-G) or FACS-sorted K562/luc GFP positive cells from xenografts (H) . (E-F,H) Percentage of parental live cell subpopulations that are: double positive for CD45 and CD235a (CD235a&CD45), positive only for CD45, or only for CD235a, or negative for both CD markers. Representative scatter plots in (E) , summary of independent co-culture experiments (n=4) in (F) . (G) Fluorescence intensity of surface CD36 protein staining in the fraction of cells positive for CD235a or CD45. Numbers correspond to fold change in shTIAR to shNEG ± ME (n=2). (H) Percent GFP and hCD45-positive cells from different xenografts (n=4) that are positive for CD235a. Two-way ANOVA test was used to for comparison of shNEG_C to other variants (@), and p=0.0046.

Article Snippet: Human chronic myeloid leukemia cell line K562 (#CCL-243) and bone marrow stroma fibroblast cell line HS-5 (#CRL-11882) were obtained from American Type Culture Collection (USA); human chronic myeloid leukemia cell line LAMA-84 (#ACC 168) was from DSMZ.

Techniques: Expressing, Cell Culture, Western Blot, Membrane, Control, Flow Cytometry, Fluorescence, Cell Differentiation, Isolation, Co-Culture Assay, Staining, Comparison

Journal: bioRxiv

Article Title: Coupling Mechanical Regulation with Biochemical Reaction-Diffusion Circuits Yields Robust Self-Organized Pattern Formation

doi: 10.64898/2026.05.23.727407

Figure Lengend Snippet: Cell A: We engineered L929 cells to express anti-CD19 synNotch that induces membrane-tethered GFP ligand and mCherry reporter . The expression level of mCherry reporter was measured with or without stimulation by CD19 ligand -expressing K562 cells (K562/CD19) using flow cytometry. Cell B: We engineered L929 cells to express anti-GFP LaG17 synNotch that induces CD19 ligand , BFP reporter and rtTA. The expression level of BFP reporter was measured with or without synNotch stimulation by GFP ligand -expressing K562 cells (K562/GFP) using flow cytometry. Cell B was further engineered to induce the expression of GFP inhibitor and IFP reporter by rtTA in the presence of Dox. The induction levels of IFP reporter in activated Cell B were tunable with variable Dox concentrations.

Article Snippet: Human erythroleukemic cell line K562 (ATCC, #CCL-243) was maintained with DMEM/10%FBS in non-treat culture dish.

Techniques: Membrane, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Coupling Mechanical Regulation with Biochemical Reaction-Diffusion Circuits Yields Robust Self-Organized Pattern Formation

doi: 10.64898/2026.05.23.727407

Figure Lengend Snippet: (A) Cell A-iPcad: Cell A was further engineered to induce P-cadherin and BFP reporter in the downstream of anti-CD19 synNotch. The expression level of both mCherry reporter and BFP reporter were measured with or without stimulation by K562/CD19 using flow cytometry. Cell B-iPcad: Cell B was further engineered to induce P-cadherin with mCherry reporter in the downstream of anti-GFP LaG17 synNotch. The expression level of both BFP reporter and mCherry reporter were measured with or without synNotch stimulation by K562/GFP using flow cytometry. The induction levels of IFP reporter in Cell B-iPcad show similar tunability to Cell B in the presence of variable Dox concentrations. (B) Comparable adhesion induction in Cell A-iPcad cells and Cell B-iPcad. 20000 cells of each Cell A-iPcad and Cell B-iPcad were cocultured in the absence of Dox, leading to activation through positive feedback and formation of large cell aggregates. By labeling Cell A-iPcad with CellTrace Far red dye, well-mixed distributions of Cell A-iPcad cells in the aggregates were visualized, suggesting that both cell types induced similar levels of P-cadherin expression when activated.

Article Snippet: Human erythroleukemic cell line K562 (ATCC, #CCL-243) was maintained with DMEM/10%FBS in non-treat culture dish.

Techniques: Expressing, Flow Cytometry, Activation Assay, Labeling

Journal: PLOS One

Article Title: BaEV-pseudotyped lentiviral vectors enable stable CAR expression and cytotoxic function in NK cells

doi: 10.1371/journal.pone.0348674

Figure Lengend Snippet: (A) Schematic representation of the transfer vector used in this study. (B) Average virus titer (viral genome copies/mL) of each LV (n = 5). (C) A total of 2.5x10 5 NK92 cells were transduced with each type of lentiviral particle using 2.32x10 9 viral genome copies. The CAR expression level of each CAR-NK92 cell group was assessed by CD19 scFv-positive cell percentage on days 3, 7, and 14 post-transduction (day 3, n = 4; days 7 and 14, n = 5). (D) Cytotoxic activity of untransduced (UTD) and CAR-NK92 cells against K562 and Nalm-6. Cells were co-cultured for 4 hours at indicated E:T ratios (n = 4). (E) qRT-PCR analysis of the relative gene expression of CAR (n = 3). Data are shown as relative CD19 scFv gene expression using human GAPDH as a reference gene with analysis by the 2-ΔΔC T algorithm. (F) Gel electrophoresis analysis of CD19 scFv and human GAPDH amplified from genomic DNA of both UTD and CAR-NK92 cells at days 7 and 14 after transduction. DNA was extracted as total cellular genomic DNA, which may also contain non-integrated residual viral genomes. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test (C), and one-way ANOVA with Tukey’s multiple comparisons test (B, E). *, p < 0.05; **, p < 0.01; ****, p < 0.0001; ns, non-significant.

Article Snippet: The human cell lines K562 (chronic myeloid leukemia), Nalm-6 (B cell lineage acute lymphoblastic leukemia), and NK92 (NK cell lymphoma) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Plasmid Preparation, Virus, Transduction, Expressing, Activity Assay, Cell Culture, Quantitative RT-PCR, Gene Expression, Nucleic Acid Electrophoresis, Amplification, Standard Deviation

Journal: PLOS One

Article Title: BaEV-pseudotyped lentiviral vectors enable stable CAR expression and cytotoxic function in NK cells

doi: 10.1371/journal.pone.0348674

Figure Lengend Snippet: (A) Schematic representation of 19BBz and 19BBz-IL15 CAR vectors. (B) CAR expression by NK92 cells transduced with 19BBz CAR or 19BBz-IL15 CAR using BaEV-LVs was measured on day 7 post-transduction (n = 6). (C) IL-15 secretion by both UTD and CAR-NK92 cells cultured without exogenous cytokine for 24 and 48 hours, measured by ELISA (n = 4). (D) Cell survival analysis of each CAR-NK92 cell group performed under cytokine starvation condition. The absolute cell numbers were counted on days 0, 4, and 7 of culture using counting beads with P.I. staining (n = 3). (E) Cytotoxic activity of UTD and CAR-NK92 cells against CD19-negative K562 and CD19-positive Nalm-6 cells. Cells were co-cultured for 4 hours at indicated E:T ratios (UTD, n = 4; CAR, n = 5). (F) Long-term cytotoxicity assay of UTD and CAR-NK92 cells against K562 (left) and Nalm-6 (right) cells. Cells were co-cultured for 24, 48 and 72 hours at an E:T ratio of 0.25:1. The live target cell percentage was calculated by gating each effector and target cell type (K562, n = 3; Nalm-6, n = 6). (G, H) Cryopreserved NK92 cells were thawed and cultured overnight with 200 U/ml of IL-2. At least 70% cell viability was confirmed prior to post-thaw experiments. (G) CAR expression by each CAR-NK92 cell group detected before (Fresh) and after (Thaw) cryopreservation (n = 3). (H) Post-thaw cytotoxic activity of UTD and CAR NK-92 cells against K562 and Nalm-6. Cells were co-cultured for 4 hours at indicated E:T ratios (n = 4). Data are presented as mean ± SD. Statistical analysis was performed using a one-way ANOVA with Tukey’s multiple comparisons test (C), two-way ANOVA with Tukey’s multiple comparisons test (F), and a two-tailed unpaired Student’s t-test (D,G); *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, non-significant.

Article Snippet: The human cell lines K562 (chronic myeloid leukemia), Nalm-6 (B cell lineage acute lymphoblastic leukemia), and NK92 (NK cell lymphoma) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Transduction, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Activity Assay, Cytotoxicity Assay, Two Tailed Test

Journal: PLOS One

Article Title: BaEV-pseudotyped lentiviral vectors enable stable CAR expression and cytotoxic function in NK cells

doi: 10.1371/journal.pone.0348674

Figure Lengend Snippet: (A) Cell proliferation of eNK cells transduced with 19BBz CAR using BaEV-LVs. A total of 2.5x10 5 eNK cells were transduced with each type of lentiviral particle using 1.16x10 9 viral genome copies (n = 4). (B) CAR expression by eNK cells transduced with 19BBz CAR or 19BBz-IL15 CAR using BaEV-LVs was measured on day 7 post-transduction (n = 8). Each dot represents different donors or replicate experiments for the same donor. (C) Cytotoxicity of UTD and CAR-eNK cells against CD19-negative K562 and CD19-positive Nalm-6 cells. Cells were co-cultured for 4 hours at indicated E:T ratios (n = 3). Cells were used after 7 days of transduction. (D) Long-term cytotoxicity assay of UTD and CAR-eNK cells against K562 (left) and Nalm-6 (right) cells. Cells were co-cultured for 24, 48 and 72 hours at an E:T ratio of 0.25:1. The live target cell percentage was calculated by gating each effector and target cell type (n = 7). Each dot represents different donors or replicate experiments for the same donor. (E, F) Cryopreserved eNK cells were thawed and cultured overnight with 200 U/ml of IL-2. At least 70% cell viability was confirmed prior to post-thaw experiments. (E) CAR expression by each CAR-eNK cell group detected before (Fresh) and after (Thaw) cryopreservation (n = 4). (F) Post-thaw cytotoxic activity of UTD and CAR-eNK cells against K562 and Nalm-6 cells. Cells were co-cultured for 4 hours at indicated E:T ratios (n = 3). Data are presented as mean ± SD. Statistical analysis was performed using a two-way ANOVA with Tukey’s multiple comparisons test (D) and two-tailed unpaired Student’s t-test (E); *, p < 0.05; **, p < 0.01; ns, non-significant.

Article Snippet: The human cell lines K562 (chronic myeloid leukemia), Nalm-6 (B cell lineage acute lymphoblastic leukemia), and NK92 (NK cell lymphoma) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Transduction, Expressing, Cell Culture, Cytotoxicity Assay, Activity Assay, Two Tailed Test