k562  (ATCC)

Journal: NAR Cancer

Article Title: RNA kinetics influence the response to transcriptional perturbation in leukaemia cell lines

doi: 10.1093/narcan/zcae039

Figure Lengend Snippet: Increased c-MYC RNA stability does not rescue biological and transcriptional effects of transcriptional inhibitors. ( A ) Schematic of CRISPR/Cas9 -homology directed repair (HDR) used to endogenously swap the c-MYC 3′ untranslated region (3′UTR) for the HPRT1 3′UTR in the K562 cell line. ( B ) Normalized expression of (left) c-MYC and (right) HPRT1 transcripts following the addition of ACTD at indicated time points as measured by quantitative real time PCR (qRT-PCR). Values are mean with error bars representing standard deviation (sd) of three biological replicates from three separate single cell clones. Data was analysed using a two-way ANOVA. **** and ns : P -value < 0.0001 and not significant, respectively. ( C ) (Left) Total gene expression of reads mapping across c-MYC and dsGFP sequences with indicated treatments and cell lines after 6 h. (Right) c-MYC intron expression with indicated treatments and cell lines after 6 h. Experiment was performed in technical duplicate. Data was analysed using edgeR. **** and ***: adjusted P -value < 0.0001 and 0.001, respectively. ( D ) Western blot of (top) c-MYC, (middle) GFP and (bottom) ACTIN protein with indicated treatments and cell lines after 6 h. Values indicated are protein levels normalized to ACTIN and DMSO controls. Image is representative of three biological replicates. Summary data is indicated in . ( E ) Scatter plot of significance and difference in total gene expression with (left) CDK9i and (right) ACTD treatment between c-MYC -control and -HPRT1 3′UTR cell lines. ( F ) Total cell number over time of indicated cell lines treated with increasing concentrations of (top) CDK9i and (bottom) ACTD. Values are mean with error bars representing standard deviation (sd) of three biological replicates. Data was analysed using a two-way ANOVA. ****, ***, ** and ns : P -Value < 0.0001, 0.001, 0.01 and not significant, respectively. logFC: log 2 fold change.

Article Snippet: Parental K562 cells (5e + 05) were washed in Phosphate Buffered Saline (PBS) twice and resuspended in Cell Line Nucleofector solution SF (16.4 ul) with Supplement (3.6 ul) (SF Cell Line 4D-nucleofector X Kit, Lonza, V4XC-2032).

Techniques: CRISPR, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation, Clone Assay, Western Blot, Control

Journal: NAR Cancer

Article Title: RNA kinetics influence the response to transcriptional perturbation in leukaemia cell lines

doi: 10.1093/narcan/zcae039

Figure Lengend Snippet: Increased c-MYC RNA stability does not rescue biological and transcriptional effects of transcriptional inhibitors. ( A ) Schematic of CRISPR/Cas9 -homology directed repair (HDR) used to endogenously swap the c-MYC 3′ untranslated region (3′UTR) for the HPRT1 3′UTR in the K562 cell line. ( B ) Normalized expression of (left) c-MYC and (right) HPRT1 transcripts following the addition of ACTD at indicated time points as measured by quantitative real time PCR (qRT-PCR). Values are mean with error bars representing standard deviation (sd) of three biological replicates from three separate single cell clones. Data was analysed using a two-way ANOVA. **** and ns : P -value < 0.0001 and not significant, respectively. ( C ) (Left) Total gene expression of reads mapping across c-MYC and dsGFP sequences with indicated treatments and cell lines after 6 h. (Right) c-MYC intron expression with indicated treatments and cell lines after 6 h. Experiment was performed in technical duplicate. Data was analysed using edgeR. **** and ***: adjusted P -value < 0.0001 and 0.001, respectively. ( D ) Western blot of (top) c-MYC, (middle) GFP and (bottom) ACTIN protein with indicated treatments and cell lines after 6 h. Values indicated are protein levels normalized to ACTIN and DMSO controls. Image is representative of three biological replicates. Summary data is indicated in . ( E ) Scatter plot of significance and difference in total gene expression with (left) CDK9i and (right) ACTD treatment between c-MYC -control and -HPRT1 3′UTR cell lines. ( F ) Total cell number over time of indicated cell lines treated with increasing concentrations of (top) CDK9i and (bottom) ACTD. Values are mean with error bars representing standard deviation (sd) of three biological replicates. Data was analysed using a two-way ANOVA. ****, ***, ** and ns : P -Value < 0.0001, 0.001, 0.01 and not significant, respectively. logFC: log 2 fold change.

Article Snippet: K562 CML parental and HDR-edited and THP-1 AML parental and inducible knockout cells were cultured in Roswell Park Memorial Institute (RPMI) medium (Thermo Fisher Scientific, Waltham, MA, USA, 11875093) containing 10% (v/v) heat-inactivated fetal bovine serum (HI-FBS; Thermo Fisher Scientific, 10099), penicillin (100 U/ml), streptomycin (100 μg/ml) (Thermo Fisher Scientific, 15140122) and 2 mM GlutaMAX (Thermo Fisher Scientific, 35050061) at 37°C and 5% carbon dioxide.

Techniques: CRISPR, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation, Clone Assay, Western Blot, Control

Journal: Cell Transplantation

Article Title: Evaluating Predictors of Quality in Liver NK Cells Among Deceased Donors

doi: 10.1177/09636897241283289

Figure Lengend Snippet: The phenotype and cytotoxicity of NK cells. (A) Compared the activation markers on NK cells between deceased donors and healthy volunteers. (B) The correlation between each activation marker and the cytotoxicity of NK cells against HepG2 tumor cells. (C) The correlation between TRAIL and other activation markers. (D) The correlation between each activation marker and the cytotoxicity of NK cells against K562 tumor cells. NK: natural killer; TRAIL: TNF-related apoptosis-inducing ligand.

Article Snippet: K562, a human chronic myelogenous leukemia cell line (#CCl-243, ATCC, Manassas, VA, USA) that lacks the TRAIL-death receptors (DR4/5), was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal calf serum (Mediatech, Inc, Manassas, VA, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Waltham, MA, USA) at 37°C in 5% CO 2 .

Techniques: Activation Assay, Marker

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: INTASYL self-delivering RNAi decreases TIGIT expression, enhancing NK cell cytotoxicity: a potential application to increase the efficacy of NK adoptive cell therapy against cancer

doi: 10.1007/s00262-024-03835-x

Figure Lengend Snippet: Increased target cell killing and IFN- γ and granzyme B release in the KHYG-1 cell line. Treatment of KHYG-1 cells with PH-804 resulted in increased K562 cell killing (A) and IFN- γ ( B.) and granzyme B release ( C.) by KHYG-1 cells following 4 h of coculture with K562 cells. n = 3 per experiment. The statistical significance of differences in group means was compared by one-way ANOVA and Tukey’s multiple comparisons post hoc tests. ** p < 0.01; * p < 0.05.

Article Snippet: The cells were then resuspended in fresh culture media and incubated at 37 °C for 72 h. Following the remainder of the incubation period, the cytotoxicity of KHYG-1 against K562 cells was evaluated using a DELFIA cytotoxicity assay (PerkinElmer).

Techniques:

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: INTASYL self-delivering RNAi decreases TIGIT expression, enhancing NK cell cytotoxicity: a potential application to increase the efficacy of NK adoptive cell therapy against cancer

doi: 10.1007/s00262-024-03835-x

Figure Lengend Snippet: Increased target cell killing, IFN- γ , and granzyme B release in PH-804-treated primary NK cells. Treatment of primary NK cells with PH-804 resulted in increased K562 cell killing (A) and IFN- γ ( B.) and granzyme B release ( C .) following 4 h of coculture with K562 cells. n = 3 per experiment. The statistical significance of differences in group means was compared by one-way ANOVA and Tukey’s multiple comparisons post hoc tests. **** p < 0.0001; ** p < 0.01; * p < 0.05

Article Snippet: The cells were then resuspended in fresh culture media and incubated at 37 °C for 72 h. Following the remainder of the incubation period, the cytotoxicity of KHYG-1 against K562 cells was evaluated using a DELFIA cytotoxicity assay (PerkinElmer).

Techniques:

Journal: bioRxiv

Article Title: Advancing Adoptive Cell Therapy: Optimized Expansion of Adaptive NK Cells for Solid Tumors

doi: 10.1101/2024.10.02.616358

Figure Lengend Snippet: Expansion analysis of NK cells and adaptive NK cells upon tumor antigen stimulation. a . Protocol procedure for the selective expansion of aNK cells. Figure created with BioRender.com . b . Frequency of NK and T cells of live cells as well as cell numbers of NK cells during the expansion. c . Expansion analysis by flow cytometry of NK cells and T cells on days 0-21 from a representative HD. Gating was performed on viable CD45 + cells and subsequently on CD3 - CD56 + for NK cells and CD3 + /CD56 - for T cells. d . Frequency of live cells and cell numbers of aNK and cNK cells furing the expansion. In all cases, HD-derived PBMCs ( n=9 ), GBM TILs ( n=6 ) and IPLA-OVAC TILs ( n=4 ) were used as samples, and cells were expanded under IL-15+IL-21 stimulation together with K562/K562E feeder cells with or without overnight exposure to UL18 lysate (for GBM TILs) or TYK-nu lysate (for OVAC TILs). Data is presented as mean ± standard error of the mean (SEM), and statistical differences were rested using multiple paired t-tests, paired t-tests when comparing two groups, and multiple Wilcoxon test when analyzing the frequency and cell number of aNK cells (not normally distributed); *p<0,05, **p<0,01.

Article Snippet: Feeder cells K562 (A gift from Kärres group, original source) and K562E transfected with HLA-E*0101 (obtained from Dr. Michaëlesson’s laboratory, Karolinska Institutet) were grown in complete medium (CM), consisting of RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Thermo Fisher), 1% penicillin/streptomycin (100U/mL penicillin and 100 µg/mL streptomycin, Nordic Biolabs), and supplemented with 1mg/mL geneticin (Thermo Fisher).

Techniques: Flow Cytometry, Derivative Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: INTASYL self-delivering RNAi decreases TIGIT expression, enhancing NK cell cytotoxicity: a potential application to increase the efficacy of NK adoptive cell therapy against cancer

doi: 10.1007/s00262-024-03835-x

Figure Lengend Snippet: Increased target cell killing and IFN- γ and granzyme B release in the KHYG-1 cell line. Treatment of KHYG-1 cells with PH-804 resulted in increased K562 cell killing (A) and IFN- γ ( B.) and granzyme B release ( C.) by KHYG-1 cells following 4 h of coculture with K562 cells. n = 3 per experiment. The statistical significance of differences in group means was compared by one-way ANOVA and Tukey’s multiple comparisons post hoc tests. ** p < 0.01; * p < 0.05.

Article Snippet: The chronic myelogenous leukemia (CML) cell line K562 (ATCC) was cultured in Iscove’s modified Dulbecco’s medium (IMDM; Corning) supplemented with 10% FBS and 1% penicillin/streptomycin.

Techniques:

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: INTASYL self-delivering RNAi decreases TIGIT expression, enhancing NK cell cytotoxicity: a potential application to increase the efficacy of NK adoptive cell therapy against cancer

doi: 10.1007/s00262-024-03835-x

Figure Lengend Snippet: Increased target cell killing, IFN- γ , and granzyme B release in PH-804-treated primary NK cells. Treatment of primary NK cells with PH-804 resulted in increased K562 cell killing (A) and IFN- γ ( B.) and granzyme B release ( C .) following 4 h of coculture with K562 cells. n = 3 per experiment. The statistical significance of differences in group means was compared by one-way ANOVA and Tukey’s multiple comparisons post hoc tests. **** p < 0.0001; ** p < 0.01; * p < 0.05

Article Snippet: The chronic myelogenous leukemia (CML) cell line K562 (ATCC) was cultured in Iscove’s modified Dulbecco’s medium (IMDM; Corning) supplemented with 10% FBS and 1% penicillin/streptomycin.

Techniques:

Journal: bioRxiv

Article Title: Fourth generation CAR Tregs with PDCD1 -driven IL-10 have enhanced suppressive function

doi: 10.1101/2024.10.01.616177

Figure Lengend Snippet: ( A ) Schematic diagram of the second-generation HLA-A2-specific CAR (A2-CAR). ( B ) Schematic diagram of the PDCD1 locus and HDR templates. ( C ) Timeline for generating gene-edited A2-CAR Tregs. ( D ) Representative (left) and pooled (right) phenotypic characterisation of Tregs on day 13, gated on live CD4 + cells. Representative plots include % cells gated. ( E ) PD1 expression in A2-CAR Tregs following stimulation with HLA-A2 + PD-L1 − K562 cells, with MFI values included. Gated on live CD4 + cMyc + tNGFR + and tCD19 + cells, where appropriate. Averaged data are mean + SEM with connected series representing individual subjects (n=3-9).

Article Snippet: K562 cells (ATCC) were cultured in RPMI supplemented with 10% FBS, 100 U/mL penicillin-streptomycin and 2 mM GlutaMAX™.

Techniques: Expressing

Journal: bioRxiv

Article Title: Fourth generation CAR Tregs with PDCD1 -driven IL-10 have enhanced suppressive function

doi: 10.1101/2024.10.01.616177

Figure Lengend Snippet: ( A ) Expression of activation markers on Tregs following 48-hour co-culture with HLA-A2 + PD-L1 + K562 cells. Gated on live CD4 + cMyc + tNGFR + cells and tCD19 + cells, where appropriate. ( B-D ) A2-CAR Tregs were intravenously co-administered with autologous PBMCs into transgenic A2-NSG mice. Mice were euthanized after 21-days. ( B ) Schematic diagram of model. ( C ) Expression of FOXP3 and Helios in hCD45 + mCD45 − hCD4 + cMyc + Tregs following euthanasia. Gates were set on hCD4 + cells that engrafted into mice reconstituted with PBMCs alone. ( D ) TSDR methylation of hCD45 + mCD45 − hCD4 + cMyc + Tregs isolated from splenocytes by cell sorting. Averaged data are mean + SEM with each connected series representing an individual subject (n=3-5). Statistical significance was determined using paired two-tailed Student’s t-tests with p-values shown.

Article Snippet: K562 cells (ATCC) were cultured in RPMI supplemented with 10% FBS, 100 U/mL penicillin-streptomycin and 2 mM GlutaMAX™.

Techniques: Expressing, Activation Assay, Co-Culture Assay, Transgenic Assay, Methylation, Isolation, FACS, Two Tailed Test

Journal: bioRxiv

Article Title: Fourth generation CAR Tregs with PDCD1 -driven IL-10 have enhanced suppressive function

doi: 10.1101/2024.10.01.616177

Figure Lengend Snippet: ( A ) A2-CAR Treg and Tr1 cytokine secretion following 72-hour co-culture with blank or HLA-A2 + PD-L1 − K562 cells. ( B ) Doughnut plot showing the amount of IL-10 secreted (red) as a proportion of 11 cytokines analysed. ( C ) A2-CAR Treg and Tr1 cytokine secretion profile following co-culture with HLA-A2 + PD-L1 − K562 cells. Every 24-hours, culture supernatants were harvested, the cells were washed twice with PBS and re-cultured in fresh media with IL-2. ( D ) qPCR analysis of A2-CAR Treg IL10 expression following co-culture with HLA-A2 + PD-L1 − K562 cells. qPCRs were performed using primers that amplified mRNA corresponding to total IL10 (left), endogenous IL10 (middle) and exogenous IL10 (right). Schematic diagrams showing primer recognition sites are provided. CDS = coding sequence. Averaged data are mean + SEM with each connected series representing an individual subject (n=3-5).

Article Snippet: K562 cells (ATCC) were cultured in RPMI supplemented with 10% FBS, 100 U/mL penicillin-streptomycin and 2 mM GlutaMAX™.

Techniques: Co-Culture Assay, Cell Culture, Expressing, Amplification, Sequencing

Journal: bioRxiv

Article Title: Fourth generation CAR Tregs with PDCD1 -driven IL-10 have enhanced suppressive function

doi: 10.1101/2024.10.01.616177

Figure Lengend Snippet: A2-CAR Treg and Tr1 cytokine secretion timecourse following co-culture with HLA-A2 + PD-L1 − K562 cells. Every 24-hours, culture supernatants were harvested, the cells were washed twice with PBS and re-cultured in fresh media with IL-2. Data are average of 4 subjects. ND = not detected.

Article Snippet: K562 cells (ATCC) were cultured in RPMI supplemented with 10% FBS, 100 U/mL penicillin-streptomycin and 2 mM GlutaMAX™.

Techniques: Co-Culture Assay, Cell Culture