k562 human chronic myelogenous leukemia cell line Search Results


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    ATCC human chronic myelogenous leukemia cell line k562
    hCGBP fails to bind to methylated CpG motifs. EMSA was performed as described in Materials and Methods, using either the CpG-pos or CG11 oligonucleotide as the probe. Purified histidine-tagged hCGBP fusion protein (720-bp cDNA fragment) (0.5 μg) or 3 μg of heparin-fractionated nuclear extract derived from <t>K562</t> cells was added where indicated. Probes were methylated by Sss I methylase as described in Materials and Methods. The arrows indicate the positions of retarded EMSA complexes.
    Human Chronic Myelogenous Leukemia Cell Line K562, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore human chronic myelogenous leukemia cell line k562
    Influence of the tested inhibitors – TOS-1, TOS-2 and axitinib on 2-HG production in the <t>K562</t> ( A ) and U-251 cells ( B ). Data were analyzed using a one-way ANOVA with Bonferroni’s post-hoc test: ** p
    Human Chronic Myelogenous Leukemia Cell Line K562, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    LGC Standards GmbH human chronic myelogenous leukemia cell line k562
    Influence of the tested inhibitors – TOS-1, TOS-2 and axitinib on 2-HG production in the <t>K562</t> ( A ) and U-251 cells ( B ). Data were analyzed using a one-way ANOVA with Bonferroni’s post-hoc test: ** p
    Human Chronic Myelogenous Leukemia Cell Line K562, supplied by LGC Standards GmbH, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Pasteur Institute cell culture human chronic myelogenic leukemia cell line k562
    Dose- and time-dependent inhibition of <t>K562</t> cell growth by the lycopene in the free (a) and encapsulated (b) forms. The cells were incubated with increasing concentration of lycopene in the free and encapsulated forms and then the cell survival was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Data were expressed as mean ± standard deviation from three independent experiments (* P
    Cell Culture Human Chronic Myelogenic Leukemia Cell Line K562, supplied by Pasteur Institute, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Glaxo Smith human chronic myelogenous leukemia k562 derived k562 a2 cell line
    Dose- and time-dependent inhibition of <t>K562</t> cell growth by the lycopene in the free (a) and encapsulated (b) forms. The cells were incubated with increasing concentration of lycopene in the free and encapsulated forms and then the cell survival was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Data were expressed as mean ± standard deviation from three independent experiments (* P
    Human Chronic Myelogenous Leukemia K562 Derived K562 A2 Cell Line, supplied by Glaxo Smith, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GE Healthcare human chronic myeloid leukemia
    Dose- and time-dependent inhibition of <t>K562</t> cell growth by the lycopene in the free (a) and encapsulated (b) forms. The cells were incubated with increasing concentration of lycopene in the free and encapsulated forms and then the cell survival was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Data were expressed as mean ± standard deviation from three independent experiments (* P
    Human Chronic Myeloid Leukemia, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hCGBP fails to bind to methylated CpG motifs. EMSA was performed as described in Materials and Methods, using either the CpG-pos or CG11 oligonucleotide as the probe. Purified histidine-tagged hCGBP fusion protein (720-bp cDNA fragment) (0.5 μg) or 3 μg of heparin-fractionated nuclear extract derived from K562 cells was added where indicated. Probes were methylated by Sss I methylase as described in Materials and Methods. The arrows indicate the positions of retarded EMSA complexes.

    Journal: Molecular and Cellular Biology

    Article Title: Cloning of a Mammalian Transcriptional Activator That Binds Unmethylated CpG Motifs and Shares a CXXC Domain with DNA Methyltransferase, Human Trithorax, and Methyl-CpG Binding Domain Protein 1

    doi:

    Figure Lengend Snippet: hCGBP fails to bind to methylated CpG motifs. EMSA was performed as described in Materials and Methods, using either the CpG-pos or CG11 oligonucleotide as the probe. Purified histidine-tagged hCGBP fusion protein (720-bp cDNA fragment) (0.5 μg) or 3 μg of heparin-fractionated nuclear extract derived from K562 cells was added where indicated. Probes were methylated by Sss I methylase as described in Materials and Methods. The arrows indicate the positions of retarded EMSA complexes.

    Article Snippet: The human chronic myelogenous leukemia cell line K562, human erythroleukemia cell line HEL, and the human cervical carcinoma epithelial cell line HeLa were obtained from the American Type Culture Collection (Manassas, Va.).

    Techniques: Methylation, Purification, Derivative Assay

    Assessment of hCGBP antiserum and expression construct. (A) Chicken antiserum raised against the GST-hCGBP fusion protein (720-bp cDNA fragment) disrupts the histidine-tagged hCGBP EMSA complexes. EMSA was performed as described in Materials and Methods, using the CpG-pos probe and purified histidine-tagged hCGBP fusion protein. One microliter of hCGBP or preimmune serum was added to each of the indicated samples. Arrows indicate positions of retarded EMSA complexes. (B) Rabbit hCGBP antiserum detects an 88-kDa protein in K562 nuclear extract that is overexpressed upon transfection with the full-length hCGBP cDNA expression vector. Transfections and Western blot analysis were performed as described in Materials and Methods. pcDNA3.1 denotes the parental expression vector. The arrow indicates the 88-kDa hCGBP protein. Positions of protein standards are shown on the right. (C) The full-length hCGBP cDNA produces an 88-kDa protein following in vitro transcription-translation. In vitro expression and electrophoresis of hCGBP were performed as described in Materials and Methods, using either the T3 (antisense) or T7 (sense) promoter. The arrow denotes the 88-kDa hCGBP protein. Protein standards are shown on the right.

    Journal: Molecular and Cellular Biology

    Article Title: Cloning of a Mammalian Transcriptional Activator That Binds Unmethylated CpG Motifs and Shares a CXXC Domain with DNA Methyltransferase, Human Trithorax, and Methyl-CpG Binding Domain Protein 1

    doi:

    Figure Lengend Snippet: Assessment of hCGBP antiserum and expression construct. (A) Chicken antiserum raised against the GST-hCGBP fusion protein (720-bp cDNA fragment) disrupts the histidine-tagged hCGBP EMSA complexes. EMSA was performed as described in Materials and Methods, using the CpG-pos probe and purified histidine-tagged hCGBP fusion protein. One microliter of hCGBP or preimmune serum was added to each of the indicated samples. Arrows indicate positions of retarded EMSA complexes. (B) Rabbit hCGBP antiserum detects an 88-kDa protein in K562 nuclear extract that is overexpressed upon transfection with the full-length hCGBP cDNA expression vector. Transfections and Western blot analysis were performed as described in Materials and Methods. pcDNA3.1 denotes the parental expression vector. The arrow indicates the 88-kDa hCGBP protein. Positions of protein standards are shown on the right. (C) The full-length hCGBP cDNA produces an 88-kDa protein following in vitro transcription-translation. In vitro expression and electrophoresis of hCGBP were performed as described in Materials and Methods, using either the T3 (antisense) or T7 (sense) promoter. The arrow denotes the 88-kDa hCGBP protein. Protein standards are shown on the right.

    Article Snippet: The human chronic myelogenous leukemia cell line K562, human erythroleukemia cell line HEL, and the human cervical carcinoma epithelial cell line HeLa were obtained from the American Type Culture Collection (Manassas, Va.).

    Techniques: Expressing, Construct, Purification, Transfection, Plasmid Preparation, Western Blot, In Vitro, Electrophoresis

    BSN inhibits constitutively active STAT3 in A549 cells (A) The chemical structure of brassinin (BSN). (B) U266, DU145, A549, K562, and SCC4 cells (1 × 10 6 cells/well) were treated with 300 μM of BSN for 4 h. Whole-cell extracts were prepared and immunoblotted with antibodies for phospho-STAT3 (Tyr705) and STAT3. (C) A549, H460, and PC-9 cells (1 × 10 6 cells/well) were treated with 300 μM of BSN for 4 h. Whole-cell extracts were prepared and immunoblotted with antibodies for phospho-STAT3 (Tyr705) and STAT3. (D) A549 cells (1 × 10 6 cells/well) were treated with the indicated concentrations of BSN for 4 h. Whole-cell extracts were prepared and immunoblotted with antibodies for phospho-STAT3 (Tyr705), phospho-STAT3 (Ser727), and STAT3. (E) A549 cells (1 × 10 6 cells/well) were treated with the indicated concentrations of BSN for 4 h and analyzed for nuclear STAT3 levels by EMSA. (F) BSN causes inhibition of translocation of STAT3 to the nucleus. A549 cells (4 × 10 4 cells/well) were incubated with or without 300 μM BSN for 4h and then analyzed for the intracellular distribution of STAT3 by immunocytochemistry. The results shown here are representative of three independent experiments.

    Journal: Oncotarget

    Article Title: Brassinin inhibits STAT3 signaling pathway through modulation of PIAS-3 and SOCS-3 expression and sensitizes human lung cancer xenograft in nude mice to paclitaxel

    doi:

    Figure Lengend Snippet: BSN inhibits constitutively active STAT3 in A549 cells (A) The chemical structure of brassinin (BSN). (B) U266, DU145, A549, K562, and SCC4 cells (1 × 10 6 cells/well) were treated with 300 μM of BSN for 4 h. Whole-cell extracts were prepared and immunoblotted with antibodies for phospho-STAT3 (Tyr705) and STAT3. (C) A549, H460, and PC-9 cells (1 × 10 6 cells/well) were treated with 300 μM of BSN for 4 h. Whole-cell extracts were prepared and immunoblotted with antibodies for phospho-STAT3 (Tyr705) and STAT3. (D) A549 cells (1 × 10 6 cells/well) were treated with the indicated concentrations of BSN for 4 h. Whole-cell extracts were prepared and immunoblotted with antibodies for phospho-STAT3 (Tyr705), phospho-STAT3 (Ser727), and STAT3. (E) A549 cells (1 × 10 6 cells/well) were treated with the indicated concentrations of BSN for 4 h and analyzed for nuclear STAT3 levels by EMSA. (F) BSN causes inhibition of translocation of STAT3 to the nucleus. A549 cells (4 × 10 4 cells/well) were incubated with or without 300 μM BSN for 4h and then analyzed for the intracellular distribution of STAT3 by immunocytochemistry. The results shown here are representative of three independent experiments.

    Article Snippet: Cell lines and culture conditions Human multiple myeloma cell lines U266, human prostate carcinoma DU145, human lung cancer cell lines A549, H1299, and H460, human chronic myelogenous leukemia cell line K562, human squamous cell carcinoma SCC4, and mouse embryonic fibroblast (MEF) were obtained from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Inhibition, Translocation Assay, Incubation, Immunocytochemistry

    DRMB assay for the analysis of base lesion removal in K562 leukemia cells and normal human PBMCs DNA lesion repair activities measured by DRMB assays for (A) THF, (B) dU, or (C) Hx and compared to a control DRMB assay with no lesion (D) .

    Journal: Oncotarget

    Article Title: DNA Repair Molecular Beacon assay: a platform for real-time functional analysis of cellular DNA repair capacity

    doi: 10.18632/oncotarget.25859

    Figure Lengend Snippet: DRMB assay for the analysis of base lesion removal in K562 leukemia cells and normal human PBMCs DNA lesion repair activities measured by DRMB assays for (A) THF, (B) dU, or (C) Hx and compared to a control DRMB assay with no lesion (D) .

    Article Snippet: The K562 human chronic myelogenous leukemia cell line was obtained from ATCC and was cultured in RPMI 1640 medium with 10% fetal bovine serum as described previously [ ].

    Techniques:

    AldeRed 588-A is a specific substrate for ALDH. ( a ) Fluorescent candidates and the ALDEFLUOR™ reagent were tested with K562 and L1210/cpa cells. The x-axis represents selected detection filters of the LSRII FACS system. ( b ) AldeRed 588-A and the ALDEFLUOR™ reagent tested with L1210/cpa and L1210 cells. ( c ) Co-staining of AldeRed 588-A and the ALDEFLUOR™ reagent with K562 and L1210/cpa cells. DEAB: diethylaminobenzaldehyde.

    Journal: Nature communications

    Article Title: A red-shifted fluorescent substrate for aldehyde dehydrogenase

    doi: 10.1038/ncomms4662

    Figure Lengend Snippet: AldeRed 588-A is a specific substrate for ALDH. ( a ) Fluorescent candidates and the ALDEFLUOR™ reagent were tested with K562 and L1210/cpa cells. The x-axis represents selected detection filters of the LSRII FACS system. ( b ) AldeRed 588-A and the ALDEFLUOR™ reagent tested with L1210/cpa and L1210 cells. ( c ) Co-staining of AldeRed 588-A and the ALDEFLUOR™ reagent with K562 and L1210/cpa cells. DEAB: diethylaminobenzaldehyde.

    Article Snippet: Cell Lines and animals The K562 human chronic myelogenous leukemia cell line was purchased from American Type Culture Collection (CLL-243™) and maintained in suspension in IMDM media supplemented with 10% FBS.

    Techniques: FACS, Staining

    PRMT1-mediated suppression of megakaryocytic differentiation is dependent on activation of the p38 MAPK. K562 cells were transiently transfected with FLAG-p38α and empty vectors and analyzed for megakaryocytic differentiation 96 h after PMA treatment ( A ). Stable cell clones (p38α KD) transfected simultaneously with two p38α shRNAs ( sh-1 and sh-2 ) were selected, and the protein levels were examined by Western blot analysis ( B, upper panel ). Luc , luciferase shRNA; GAPDH , glyceraldehyde-3-phosphate dehydrogenase. Reduced expression of p38α enhanced megakaryocytic differentiation ( B, lower panel ). Treatment with the p38 inhibitor SB203580 ( SB ) greatly enhanced megakaryocytic differentiation in both parental K562 and HA-PRMT1-expressing R2-1 and R2-3 cells when analyzed 96 h after PMA treatment ( C ). The specific inhibitor of p38 MAPK, SB203580, was added 30 min before stimulation with PMA. Adherent cells were examined by phase contrast microscopy 96 h after PMA treatment ( D, left panel ) and quantified ( D, right panel ). Transient knockdown of p38α with either p38α sh-1 or p38α sh-2 shRNAs enhanced megakaryocytic differentiation in both K562 and R2-1 cells ( E ). Ectopic expression of p38α by transient transfection suppressed megakaryocytic differentiation in both K562 and PRMT1-knockdown cells (PRMT1 KD-1 and KD-2) ( F ). Cytological changes were detected by modified Wright-Giemsa staining ( F, upper panel ) and quantified ( F, lower panel ). HA-PRMT1 was transiently expressed in stable p38α-knockdown clones (p38α KD-1 and p38α KD-2) ( G ). Ectopic expression of HA-PRMT1 could no longer suppress megakaryocytic differentiation in p38α-deficient cells. Cells stably transfected with the luciferase shRNA ( Luc ) were used as a negative control. All experiments were performed at least three times, and data are presented as means ± S.E.; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Protein-arginine Methyltransferase 1 Suppresses Megakaryocytic Differentiation via Modulation of the p38 MAPK Pathway in K562 Cells *

    doi: 10.1074/jbc.M109.092411

    Figure Lengend Snippet: PRMT1-mediated suppression of megakaryocytic differentiation is dependent on activation of the p38 MAPK. K562 cells were transiently transfected with FLAG-p38α and empty vectors and analyzed for megakaryocytic differentiation 96 h after PMA treatment ( A ). Stable cell clones (p38α KD) transfected simultaneously with two p38α shRNAs ( sh-1 and sh-2 ) were selected, and the protein levels were examined by Western blot analysis ( B, upper panel ). Luc , luciferase shRNA; GAPDH , glyceraldehyde-3-phosphate dehydrogenase. Reduced expression of p38α enhanced megakaryocytic differentiation ( B, lower panel ). Treatment with the p38 inhibitor SB203580 ( SB ) greatly enhanced megakaryocytic differentiation in both parental K562 and HA-PRMT1-expressing R2-1 and R2-3 cells when analyzed 96 h after PMA treatment ( C ). The specific inhibitor of p38 MAPK, SB203580, was added 30 min before stimulation with PMA. Adherent cells were examined by phase contrast microscopy 96 h after PMA treatment ( D, left panel ) and quantified ( D, right panel ). Transient knockdown of p38α with either p38α sh-1 or p38α sh-2 shRNAs enhanced megakaryocytic differentiation in both K562 and R2-1 cells ( E ). Ectopic expression of p38α by transient transfection suppressed megakaryocytic differentiation in both K562 and PRMT1-knockdown cells (PRMT1 KD-1 and KD-2) ( F ). Cytological changes were detected by modified Wright-Giemsa staining ( F, upper panel ) and quantified ( F, lower panel ). HA-PRMT1 was transiently expressed in stable p38α-knockdown clones (p38α KD-1 and p38α KD-2) ( G ). Ectopic expression of HA-PRMT1 could no longer suppress megakaryocytic differentiation in p38α-deficient cells. Cells stably transfected with the luciferase shRNA ( Luc ) were used as a negative control. All experiments were performed at least three times, and data are presented as means ± S.E.; *, p

    Article Snippet: The human chronic myelogenous leukemia K562 cell line was from the American Type Culture Collection (ATCC).

    Techniques: Activation Assay, Transfection, Stable Transfection, Clone Assay, Western Blot, Luciferase, shRNA, Expressing, Microscopy, Modification, Staining, Negative Control

    Reduced levels of endogenous PRMT1 enhance PMA-induced megakaryocytic differentiation of K562 cells. KD-1 and KD-2 cell clones were stably transfected with PRMT1 shRNA ( sh-1 ). Western analysis using an anti-PRMT1 antibody showed reduced expression of PRMT1 proteins ( A, upper panel ). Megakaryocytic differentiation was analyzed 96 h after PMA treatment ( A, lower panel ). Luc , luciferase shRNA. The growth and viability of these cell clones were similar under regular culture conditions ( B ). Effects of PRMT1 knockdown were also assayed by transient transfection with a different shRNA ( sh-2 ). PRMT1 protein levels were detected by Western blot analysis ( C, upper panel ) and megakaryocytic differentiation was detected by modified Wright-Giemsa staining ( C, lower panel ). Luciferase shRNA ( Luc ) and the pSUPER empty vector ( V ) were used as controls. All experiments were performed at least three times, and data are presented as means ± S.E.; **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Protein-arginine Methyltransferase 1 Suppresses Megakaryocytic Differentiation via Modulation of the p38 MAPK Pathway in K562 Cells *

    doi: 10.1074/jbc.M109.092411

    Figure Lengend Snippet: Reduced levels of endogenous PRMT1 enhance PMA-induced megakaryocytic differentiation of K562 cells. KD-1 and KD-2 cell clones were stably transfected with PRMT1 shRNA ( sh-1 ). Western analysis using an anti-PRMT1 antibody showed reduced expression of PRMT1 proteins ( A, upper panel ). Megakaryocytic differentiation was analyzed 96 h after PMA treatment ( A, lower panel ). Luc , luciferase shRNA. The growth and viability of these cell clones were similar under regular culture conditions ( B ). Effects of PRMT1 knockdown were also assayed by transient transfection with a different shRNA ( sh-2 ). PRMT1 protein levels were detected by Western blot analysis ( C, upper panel ) and megakaryocytic differentiation was detected by modified Wright-Giemsa staining ( C, lower panel ). Luciferase shRNA ( Luc ) and the pSUPER empty vector ( V ) were used as controls. All experiments were performed at least three times, and data are presented as means ± S.E.; **, p

    Article Snippet: The human chronic myelogenous leukemia K562 cell line was from the American Type Culture Collection (ATCC).

    Techniques: Clone Assay, Stable Transfection, Transfection, shRNA, Western Blot, Expressing, Luciferase, Modification, Staining, Plasmid Preparation

    Effect of PRMT2 and PRMT5 on megakaryocytic differentiation. K562 cells were transiently transfected with pPCDNA3HA2 plasmids expressing either HA-tagged PRMT1, PRMT2, or PRMT5 proteins that were detected by Western blot ( WB ) using anti-HA antibodies ( A ). Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) was used as a loading control. Megakaryocytic differentiation was analyzed by modified Wright-Giemsa staining 96 h after PMA treatment ( B ). All experiments were performed at least three times, and data are presented as means ± S.E.; ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: Protein-arginine Methyltransferase 1 Suppresses Megakaryocytic Differentiation via Modulation of the p38 MAPK Pathway in K562 Cells *

    doi: 10.1074/jbc.M109.092411

    Figure Lengend Snippet: Effect of PRMT2 and PRMT5 on megakaryocytic differentiation. K562 cells were transiently transfected with pPCDNA3HA2 plasmids expressing either HA-tagged PRMT1, PRMT2, or PRMT5 proteins that were detected by Western blot ( WB ) using anti-HA antibodies ( A ). Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) was used as a loading control. Megakaryocytic differentiation was analyzed by modified Wright-Giemsa staining 96 h after PMA treatment ( B ). All experiments were performed at least three times, and data are presented as means ± S.E.; ***, p

    Article Snippet: The human chronic myelogenous leukemia K562 cell line was from the American Type Culture Collection (ATCC).

    Techniques: Transfection, Expressing, Western Blot, Modification, Staining

    THAP11 accelerates megakaryocytic differentiation of K562 cells induced by PMA. THAP11 lentivirus infected K562 cells (THAP11-LV) and control cells were treated with 10 nM PMA for the indicated lengths of time. CD41 + cells (A) and percentage of 4N cells (B) were analyzed using FACS. The CD61 mRNA level was measured by real-time PCR analysis (C). (D) THAP11 siRNA lentivirus infected-K562 cells or control K562 cells were treated with 10 nM PMA for 3 days. Then CD41 + cells and the percentage of 4N cells (E) were analyzed using FACS. (F) The CD61 mRNA level was measured using real-time PCR analysis.

    Journal: PLoS ONE

    Article Title: Effects of THAP11 on Erythroid Differentiation and Megakaryocytic Differentiation of K562 Cells

    doi: 10.1371/journal.pone.0091557

    Figure Lengend Snippet: THAP11 accelerates megakaryocytic differentiation of K562 cells induced by PMA. THAP11 lentivirus infected K562 cells (THAP11-LV) and control cells were treated with 10 nM PMA for the indicated lengths of time. CD41 + cells (A) and percentage of 4N cells (B) were analyzed using FACS. The CD61 mRNA level was measured by real-time PCR analysis (C). (D) THAP11 siRNA lentivirus infected-K562 cells or control K562 cells were treated with 10 nM PMA for 3 days. Then CD41 + cells and the percentage of 4N cells (E) were analyzed using FACS. (F) The CD61 mRNA level was measured using real-time PCR analysis.

    Article Snippet: Cell culture and induced differentiation Human chronic myelogenous leukemia cell line K562 cells and TF-1 cells were purchased from American Type Culture Collection (Manassas, VA).

    Techniques: Infection, FACS, Real-time Polymerase Chain Reaction

    Alteration of expression levels of several hematopoietic transcription factors in THAP11-overexpressing K562 cells. K562 cells infected with THAP11-lentiviruses (THAP11-LV) and control cells were treated with 10 nM PMA for 72 hours. Then total RNA was extracted for real-time PCR analysis. Real-time PCR results were expressed as fold induction relative to cells at day 0 and normalized to GAPDH mRNA. Each bar represented the mean ± SD for three independent experiments. The statistical difference between the samples was demonstrated as * P ≤0.05 or ** P ≤0.001.

    Journal: PLoS ONE

    Article Title: Effects of THAP11 on Erythroid Differentiation and Megakaryocytic Differentiation of K562 Cells

    doi: 10.1371/journal.pone.0091557

    Figure Lengend Snippet: Alteration of expression levels of several hematopoietic transcription factors in THAP11-overexpressing K562 cells. K562 cells infected with THAP11-lentiviruses (THAP11-LV) and control cells were treated with 10 nM PMA for 72 hours. Then total RNA was extracted for real-time PCR analysis. Real-time PCR results were expressed as fold induction relative to cells at day 0 and normalized to GAPDH mRNA. Each bar represented the mean ± SD for three independent experiments. The statistical difference between the samples was demonstrated as * P ≤0.05 or ** P ≤0.001.

    Article Snippet: Cell culture and induced differentiation Human chronic myelogenous leukemia cell line K562 cells and TF-1 cells were purchased from American Type Culture Collection (Manassas, VA).

    Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction

    THAP11 overexpression leads to inhibition of hemin induced erythroid differentiation of K562 cells. (A) THAP11 lentivirus infected K562 cells (THAP11-LV) and control cells were treated with 40 µM hemin for the indicated lengths of time and the benzidine-positive cells were counted. The pictures of cells treated with hemin for 5 days were shown in (B). (C) HBA mRNA level and (D) GPA mRNA level were detected using real-time PCR analysis. Real-time PCR results were expressed as fold induction relative to control cells at day 0 and normalized to GAPDH mRNA.

    Journal: PLoS ONE

    Article Title: Effects of THAP11 on Erythroid Differentiation and Megakaryocytic Differentiation of K562 Cells

    doi: 10.1371/journal.pone.0091557

    Figure Lengend Snippet: THAP11 overexpression leads to inhibition of hemin induced erythroid differentiation of K562 cells. (A) THAP11 lentivirus infected K562 cells (THAP11-LV) and control cells were treated with 40 µM hemin for the indicated lengths of time and the benzidine-positive cells were counted. The pictures of cells treated with hemin for 5 days were shown in (B). (C) HBA mRNA level and (D) GPA mRNA level were detected using real-time PCR analysis. Real-time PCR results were expressed as fold induction relative to control cells at day 0 and normalized to GAPDH mRNA.

    Article Snippet: Cell culture and induced differentiation Human chronic myelogenous leukemia cell line K562 cells and TF-1 cells were purchased from American Type Culture Collection (Manassas, VA).

    Techniques: Over Expression, Inhibition, Infection, Real-time Polymerase Chain Reaction

    THAP11 knockdown enhances hemin induced erythroid differentiation of K562 cells. (A) K562 cells were infected with control lentivirus or THAP11 RNAi lentivirus (siTHAP11-1 and siTHAP11-2) and then the GFP positive cells were sorted. The THAP11 expression level was detected by real-time PCR (upper panel) and Western blot analysis (lower panel). Then the cells were treated with 40 µM hemin for the indicated lengths of time. (B) Benzidine-positive cells were counted. (C) HBA and (D) GPA mRNA levels were measured using real-time PCR analysis.

    Journal: PLoS ONE

    Article Title: Effects of THAP11 on Erythroid Differentiation and Megakaryocytic Differentiation of K562 Cells

    doi: 10.1371/journal.pone.0091557

    Figure Lengend Snippet: THAP11 knockdown enhances hemin induced erythroid differentiation of K562 cells. (A) K562 cells were infected with control lentivirus or THAP11 RNAi lentivirus (siTHAP11-1 and siTHAP11-2) and then the GFP positive cells were sorted. The THAP11 expression level was detected by real-time PCR (upper panel) and Western blot analysis (lower panel). Then the cells were treated with 40 µM hemin for the indicated lengths of time. (B) Benzidine-positive cells were counted. (C) HBA and (D) GPA mRNA levels were measured using real-time PCR analysis.

    Article Snippet: Cell culture and induced differentiation Human chronic myelogenous leukemia cell line K562 cells and TF-1 cells were purchased from American Type Culture Collection (Manassas, VA).

    Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction, Western Blot

    Prostein-specific lysis and HLA-A * 0201-restricted recognition of LNCaP 1740 cells by the in vitro -generated cytotoxic effector cells. ( A ) After four rounds of stimulation activated CD8+ T cells from the three donors were cocultured with 3 × 10 3 51 Cr-labeled LNCaP 1740, 93.04A12.1 or K562 tumour cells well −1 at various effector cell (E) to target cell (T) ratios (3 : 1, 10 : 1, 30 : 1). After 4 h of incubation, chromium release was determined. ( B ) The inhibition of T-cell-mediated cytotoxicity against LNCaP 1740 cells was tested in the presence of the monoclonal anti-HLA-A2 antibody MA2.1 at an E : T ratio of 30 : 1. ( C ) Influence of androgen deprivation or supplementation on the prostein-specific lysis of LNCaP 1740 target cells. LNCaP 1740 cells were cultured for 24 h in medium containing charcoal-stipped FCS. Cells were grown for additional 48 h in the androgen-deprived medium or in the presence of 1 n M R1881 and then used as target cells in a chromium release assay at an E : T ratio of 30 : 1. All results represent the mean values of triplicate determinations, bars indicate s.e.

    Journal: British Journal of Cancer

    Article Title: Identification of an HLA-A*0201-restricted T-cell epitope derived from the prostate cancer-associated protein prostein

    doi: 10.1038/sj.bjc.6601642

    Figure Lengend Snippet: Prostein-specific lysis and HLA-A * 0201-restricted recognition of LNCaP 1740 cells by the in vitro -generated cytotoxic effector cells. ( A ) After four rounds of stimulation activated CD8+ T cells from the three donors were cocultured with 3 × 10 3 51 Cr-labeled LNCaP 1740, 93.04A12.1 or K562 tumour cells well −1 at various effector cell (E) to target cell (T) ratios (3 : 1, 10 : 1, 30 : 1). After 4 h of incubation, chromium release was determined. ( B ) The inhibition of T-cell-mediated cytotoxicity against LNCaP 1740 cells was tested in the presence of the monoclonal anti-HLA-A2 antibody MA2.1 at an E : T ratio of 30 : 1. ( C ) Influence of androgen deprivation or supplementation on the prostein-specific lysis of LNCaP 1740 target cells. LNCaP 1740 cells were cultured for 24 h in medium containing charcoal-stipped FCS. Cells were grown for additional 48 h in the androgen-deprived medium or in the presence of 1 n M R1881 and then used as target cells in a chromium release assay at an E : T ratio of 30 : 1. All results represent the mean values of triplicate determinations, bars indicate s.e.

    Article Snippet: Cell lines The PCa cell lines LNCaP 1740 and PC-3, the mutant cell line T2 and the chronic myelogenous leukemia cell line K562 (all from American Type Culture Collection, Manassas, VA, USA) were cultured according to the manufacturer's instructions.

    Techniques: Lysis, In Vitro, Generated, Labeling, Incubation, Inhibition, Cell Culture, Release Assay

    Inhibition of Pim-2 expression by specific siRNA in transcriptional level. Quantitative expression of Pim-2 mRNA in Pim-2 siRNA and control siRNA transfected cells. Pim-2 siRNA effectively silenced Pim-2 in K562, RPMI-8226, H1299 and A549 cell lines (*P

    Journal: Oncology Letters

    Article Title: Downregulation of Pim-2 induces cell cycle arrest in the G0/G1 phase via the p53-non-dependent p21 signaling pathway

    doi: 10.3892/ol.2018.7865

    Figure Lengend Snippet: Inhibition of Pim-2 expression by specific siRNA in transcriptional level. Quantitative expression of Pim-2 mRNA in Pim-2 siRNA and control siRNA transfected cells. Pim-2 siRNA effectively silenced Pim-2 in K562, RPMI-8226, H1299 and A549 cell lines (*P

    Article Snippet: K562 chronic myelogenous leukemia cell line, RPMI-8226MM cell line and H1299 and A549 non-small cell lung carcinoma cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA), and grown in RPMI-1640 medium (Boehringer, Ingelheim, Germany) supplemented with 10% heat-inactivated fetal calf serum (Boehringer), 100 µg/ml penicillin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 100 U/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.), in a humidified atmosphere (37.5°C; 5% CO2 ).

    Techniques: Inhibition, Expressing, Transfection

    Pim-2 protein expression levels in K562, RPMI-8226, H1299 and A549 cell lines. Western blot analysis of NF-κB and Pim-2 expression levels. NF-κB, nuclear factor-κB.

    Journal: Oncology Letters

    Article Title: Downregulation of Pim-2 induces cell cycle arrest in the G0/G1 phase via the p53-non-dependent p21 signaling pathway

    doi: 10.3892/ol.2018.7865

    Figure Lengend Snippet: Pim-2 protein expression levels in K562, RPMI-8226, H1299 and A549 cell lines. Western blot analysis of NF-κB and Pim-2 expression levels. NF-κB, nuclear factor-κB.

    Article Snippet: K562 chronic myelogenous leukemia cell line, RPMI-8226MM cell line and H1299 and A549 non-small cell lung carcinoma cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA), and grown in RPMI-1640 medium (Boehringer, Ingelheim, Germany) supplemented with 10% heat-inactivated fetal calf serum (Boehringer), 100 µg/ml penicillin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 100 U/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.), in a humidified atmosphere (37.5°C; 5% CO2 ).

    Techniques: Expressing, Western Blot

    Effect of inhibition of Pim-2 expression level on K562, RPMI-8226, H1299 and A549 cell proliferation. Cell proliferation was determined by cell counting kit-8 assay. Pim-2 silencing inhibited cell proliferation in the cell lines. Results are presented as the mean ± standard deviation of 3 replicates and it is representative of 3 independent experiments (*P

    Journal: Oncology Letters

    Article Title: Downregulation of Pim-2 induces cell cycle arrest in the G0/G1 phase via the p53-non-dependent p21 signaling pathway

    doi: 10.3892/ol.2018.7865

    Figure Lengend Snippet: Effect of inhibition of Pim-2 expression level on K562, RPMI-8226, H1299 and A549 cell proliferation. Cell proliferation was determined by cell counting kit-8 assay. Pim-2 silencing inhibited cell proliferation in the cell lines. Results are presented as the mean ± standard deviation of 3 replicates and it is representative of 3 independent experiments (*P

    Article Snippet: K562 chronic myelogenous leukemia cell line, RPMI-8226MM cell line and H1299 and A549 non-small cell lung carcinoma cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA), and grown in RPMI-1640 medium (Boehringer, Ingelheim, Germany) supplemented with 10% heat-inactivated fetal calf serum (Boehringer), 100 µg/ml penicillin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 100 U/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.), in a humidified atmosphere (37.5°C; 5% CO2 ).

    Techniques: Inhibition, Expressing, Cell Counting, Standard Deviation

    Effect of inhibition of Pim-2 in K562, RPMI-8226, H1299 and A549 cells on the cell cycle regulatory protein expression levels. Western blot analysis of cell cycle regulatory proteins P53, P21, CDK2, pRb and Rb in control and pim-2 silenced cells. p21 was highly expressed in all the cell lines. GAPDH was used as endogenous control. CDK2, cell dependent kinase 2; Rb, retinoblastoma; p, phosphorylated; siRNA, short interfering RNA.

    Journal: Oncology Letters

    Article Title: Downregulation of Pim-2 induces cell cycle arrest in the G0/G1 phase via the p53-non-dependent p21 signaling pathway

    doi: 10.3892/ol.2018.7865

    Figure Lengend Snippet: Effect of inhibition of Pim-2 in K562, RPMI-8226, H1299 and A549 cells on the cell cycle regulatory protein expression levels. Western blot analysis of cell cycle regulatory proteins P53, P21, CDK2, pRb and Rb in control and pim-2 silenced cells. p21 was highly expressed in all the cell lines. GAPDH was used as endogenous control. CDK2, cell dependent kinase 2; Rb, retinoblastoma; p, phosphorylated; siRNA, short interfering RNA.

    Article Snippet: K562 chronic myelogenous leukemia cell line, RPMI-8226MM cell line and H1299 and A549 non-small cell lung carcinoma cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA), and grown in RPMI-1640 medium (Boehringer, Ingelheim, Germany) supplemented with 10% heat-inactivated fetal calf serum (Boehringer), 100 µg/ml penicillin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 100 U/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.), in a humidified atmosphere (37.5°C; 5% CO2 ).

    Techniques: Inhibition, Expressing, Western Blot, Small Interfering RNA

    The result of cell cycle by flow cytometry. (A) K562 control siRNA, (B) K562 Pim-2 siRNA, (C) RPMI-8226 control siRNA, (D) RPMI-8226 Pim-2 siRNA, (E) H1299 control siRNA, (F) H1299 Pim-2 siRNA, (G) A549 control siRNA and (H) A549 Pim-2 siRNA. M1 indicated G 0 /G 1 cell cycle phase, M2 indicated G 2 /S cell cycle phase. siRNA, short interfering RNA.

    Journal: Oncology Letters

    Article Title: Downregulation of Pim-2 induces cell cycle arrest in the G0/G1 phase via the p53-non-dependent p21 signaling pathway

    doi: 10.3892/ol.2018.7865

    Figure Lengend Snippet: The result of cell cycle by flow cytometry. (A) K562 control siRNA, (B) K562 Pim-2 siRNA, (C) RPMI-8226 control siRNA, (D) RPMI-8226 Pim-2 siRNA, (E) H1299 control siRNA, (F) H1299 Pim-2 siRNA, (G) A549 control siRNA and (H) A549 Pim-2 siRNA. M1 indicated G 0 /G 1 cell cycle phase, M2 indicated G 2 /S cell cycle phase. siRNA, short interfering RNA.

    Article Snippet: K562 chronic myelogenous leukemia cell line, RPMI-8226MM cell line and H1299 and A549 non-small cell lung carcinoma cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA), and grown in RPMI-1640 medium (Boehringer, Ingelheim, Germany) supplemented with 10% heat-inactivated fetal calf serum (Boehringer), 100 µg/ml penicillin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 100 U/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.), in a humidified atmosphere (37.5°C; 5% CO2 ).

    Techniques: Flow Cytometry, Cytometry, Small Interfering RNA

    Pim-2 protein localization in K562, RPMI-8226, H1299 and A549 cell lines. Immunocytochemistry analysis was performed for localization of Pim-2. Flourescein isothiocyanate-conjugated phalloidin was used for Pim-2 (green fluorescence) and DAPI for nuclei (blue fluorescence). Pim-2 was predominantly located in the cytoplasm of all four cell lines (magnification, ×200).

    Journal: Oncology Letters

    Article Title: Downregulation of Pim-2 induces cell cycle arrest in the G0/G1 phase via the p53-non-dependent p21 signaling pathway

    doi: 10.3892/ol.2018.7865

    Figure Lengend Snippet: Pim-2 protein localization in K562, RPMI-8226, H1299 and A549 cell lines. Immunocytochemistry analysis was performed for localization of Pim-2. Flourescein isothiocyanate-conjugated phalloidin was used for Pim-2 (green fluorescence) and DAPI for nuclei (blue fluorescence). Pim-2 was predominantly located in the cytoplasm of all four cell lines (magnification, ×200).

    Article Snippet: K562 chronic myelogenous leukemia cell line, RPMI-8226MM cell line and H1299 and A549 non-small cell lung carcinoma cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA), and grown in RPMI-1640 medium (Boehringer, Ingelheim, Germany) supplemented with 10% heat-inactivated fetal calf serum (Boehringer), 100 µg/ml penicillin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 100 U/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.), in a humidified atmosphere (37.5°C; 5% CO2 ).

    Techniques: Immunocytochemistry, Fluorescence

    Effect of inhibition of NF-κB in K562, RPMI-8226, H1299 and A549 cells to Pim-2 expression level. (A) K562, (B) RPMI-8226, (C) H1299 and (D) A549. Western blot analysis of Pim-2 and NF-κB with NF-κB inhibitor at 0, 1, 2.5, 5 and 10 µΜ. Pim-2 and NF-κB expression levels were significant decreased in all the cell lines. GAPDH was used as endogenous control. NF-κB, nuclear factor-κB.

    Journal: Oncology Letters

    Article Title: Downregulation of Pim-2 induces cell cycle arrest in the G0/G1 phase via the p53-non-dependent p21 signaling pathway

    doi: 10.3892/ol.2018.7865

    Figure Lengend Snippet: Effect of inhibition of NF-κB in K562, RPMI-8226, H1299 and A549 cells to Pim-2 expression level. (A) K562, (B) RPMI-8226, (C) H1299 and (D) A549. Western blot analysis of Pim-2 and NF-κB with NF-κB inhibitor at 0, 1, 2.5, 5 and 10 µΜ. Pim-2 and NF-κB expression levels were significant decreased in all the cell lines. GAPDH was used as endogenous control. NF-κB, nuclear factor-κB.

    Article Snippet: K562 chronic myelogenous leukemia cell line, RPMI-8226MM cell line and H1299 and A549 non-small cell lung carcinoma cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA), and grown in RPMI-1640 medium (Boehringer, Ingelheim, Germany) supplemented with 10% heat-inactivated fetal calf serum (Boehringer), 100 µg/ml penicillin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 100 U/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.), in a humidified atmosphere (37.5°C; 5% CO2 ).

    Techniques: Inhibition, Expressing, Western Blot

    sCAR-PPAb activated macrophages in a K562/ADR xenograft model. BALB/c nude mice were transplanted with K562/ADR cells subcutaneously. sCAR-PPAb were injected intratumorally for 7 days at 100μg/day. PBS injection served as the control. A. Tumors were harvested and F4/80 was analyzed for macrophage infiltration by immunohistochemistry. The control showed IgG isotype staining. Arrows point to cells stained with F4/80. Bars show 200μm (400 x). B. Tumors were analyzed using a transmission electron microscope. The arrow points to cells undergoing phagocytosis. Bars show 2μm.

    Journal: PLoS ONE

    Article Title: Pinellia pedatisecta Agglutinin Targets Drug Resistant K562/ADR Leukemia Cells through Binding with Sarcolemmal Membrane Associated Protein and Enhancing Macrophage Phagocytosis

    doi: 10.1371/journal.pone.0074363

    Figure Lengend Snippet: sCAR-PPAb activated macrophages in a K562/ADR xenograft model. BALB/c nude mice were transplanted with K562/ADR cells subcutaneously. sCAR-PPAb were injected intratumorally for 7 days at 100μg/day. PBS injection served as the control. A. Tumors were harvested and F4/80 was analyzed for macrophage infiltration by immunohistochemistry. The control showed IgG isotype staining. Arrows point to cells stained with F4/80. Bars show 200μm (400 x). B. Tumors were analyzed using a transmission electron microscope. The arrow points to cells undergoing phagocytosis. Bars show 2μm.

    Article Snippet: Cells Human chronic myeloid leukemia cell line K562 was purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin solution, and 1% L-Glutamine.

    Techniques: Mouse Assay, Injection, Immunohistochemistry, Staining, Transmission Assay, Microscopy

    sCAR-PPAb binds with SLMAP. A. sCAR-PPAb combined with a CAR antibody was used in immunoprecipitation. PBS combined with CAR antibody served as the control. Precipitates were analyzed by Western blot for SLMAP. Whole cell lysis (WCL) was monitored for Actin levels. B. SLMAP levels on K562 and K562/ADR were determined by Western blot. Actin served as a loading control. C. A SLMAP antibody enhanced the phagocytosis of K562/ADR by macrophages in vitro. An IgG isotype served as the control. The portion of macrophages ingesting CFSE stained K562/ADR cells was observed under a fluorescence microscope (200 x). A significant enhancement of phagocytosis upon SLMAP antibody treatment was determined (p

    Journal: PLoS ONE

    Article Title: Pinellia pedatisecta Agglutinin Targets Drug Resistant K562/ADR Leukemia Cells through Binding with Sarcolemmal Membrane Associated Protein and Enhancing Macrophage Phagocytosis

    doi: 10.1371/journal.pone.0074363

    Figure Lengend Snippet: sCAR-PPAb binds with SLMAP. A. sCAR-PPAb combined with a CAR antibody was used in immunoprecipitation. PBS combined with CAR antibody served as the control. Precipitates were analyzed by Western blot for SLMAP. Whole cell lysis (WCL) was monitored for Actin levels. B. SLMAP levels on K562 and K562/ADR were determined by Western blot. Actin served as a loading control. C. A SLMAP antibody enhanced the phagocytosis of K562/ADR by macrophages in vitro. An IgG isotype served as the control. The portion of macrophages ingesting CFSE stained K562/ADR cells was observed under a fluorescence microscope (200 x). A significant enhancement of phagocytosis upon SLMAP antibody treatment was determined (p

    Article Snippet: Cells Human chronic myeloid leukemia cell line K562 was purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin solution, and 1% L-Glutamine.

    Techniques: Immunoprecipitation, Western Blot, Lysis, In Vitro, Staining, Fluorescence, Microscopy

    PPA preferentially recognizes drug resistant cancer cells. A. The sensitivity of K562 and K562/ADR to ADR was determined by a MTT assay. B. K562 or K562/ADR cells were treated with sCAR-PPAb combined with Ad-EGFP or PBS for 48h. The portion of EGFP positive cells was analyzed by flow cytometry. Shown is a representative from 3 separate experiments. C. K562 or K562/ADR cells treated with sCAR-PPAb combined with Ad-EGFP for 48h were examined using a fluorescence microscope. D. The sensitivity of H460 and H460/5Fu to 5Fu was determined by a MTT assay. E. H460 or H460/5Fu cells were treated with sCAR-PPAb combined with Ad-EGFP or PBS for 48h. The portion of EGFP positive cells was analyzed by flow cytometry. Shown is a representative from 3 separate experiments. F. H460 or H460/5Fu cells treated with sCAR-PPAb combined with Ad-EGFP for 48h were examined using a fluorescence microscope.

    Journal: PLoS ONE

    Article Title: Pinellia pedatisecta Agglutinin Targets Drug Resistant K562/ADR Leukemia Cells through Binding with Sarcolemmal Membrane Associated Protein and Enhancing Macrophage Phagocytosis

    doi: 10.1371/journal.pone.0074363

    Figure Lengend Snippet: PPA preferentially recognizes drug resistant cancer cells. A. The sensitivity of K562 and K562/ADR to ADR was determined by a MTT assay. B. K562 or K562/ADR cells were treated with sCAR-PPAb combined with Ad-EGFP or PBS for 48h. The portion of EGFP positive cells was analyzed by flow cytometry. Shown is a representative from 3 separate experiments. C. K562 or K562/ADR cells treated with sCAR-PPAb combined with Ad-EGFP for 48h were examined using a fluorescence microscope. D. The sensitivity of H460 and H460/5Fu to 5Fu was determined by a MTT assay. E. H460 or H460/5Fu cells were treated with sCAR-PPAb combined with Ad-EGFP or PBS for 48h. The portion of EGFP positive cells was analyzed by flow cytometry. Shown is a representative from 3 separate experiments. F. H460 or H460/5Fu cells treated with sCAR-PPAb combined with Ad-EGFP for 48h were examined using a fluorescence microscope.

    Article Snippet: Cells Human chronic myeloid leukemia cell line K562 was purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin solution, and 1% L-Glutamine.

    Techniques: MTT Assay, Flow Cytometry, Cytometry, Fluorescence, Microscopy

    sCAR-PPAb enhances the phagocytosis of K562/ADR cells by macrophages in vivo. A. sCAR-PPAb did not enhance the phagocytosis of K562 cells by macrophages. K562 cells stained with CFSE and pretreated with sCAR-PPAb or PBS were injected into the abdominal cavity in ICR mice. After 5h, cells in the abdominal cavity were then collected and stained with a primary antibody against F4/80 or IgG isotype followed by a goat anti-rat IgG-PE antibody. Cells were then analyzed by flow cytometry. Data were reported as the percent of macrophages undergoing phagocytosis, calculated by CFSE/PE double positive cells divided by all PE positive cells. Values are shown as mean ± SEM (p > 0.05, n=3). B. sCAR-PPAb enhanced the phagocytosis of K562/ADR by macrophages. Data were reported as the percent of macrophages undergoing phagocytosis, calculated by CFSE/PE double positive cells divided by all PE positive cells. Values are shown as mean ± SEM (p

    Journal: PLoS ONE

    Article Title: Pinellia pedatisecta Agglutinin Targets Drug Resistant K562/ADR Leukemia Cells through Binding with Sarcolemmal Membrane Associated Protein and Enhancing Macrophage Phagocytosis

    doi: 10.1371/journal.pone.0074363

    Figure Lengend Snippet: sCAR-PPAb enhances the phagocytosis of K562/ADR cells by macrophages in vivo. A. sCAR-PPAb did not enhance the phagocytosis of K562 cells by macrophages. K562 cells stained with CFSE and pretreated with sCAR-PPAb or PBS were injected into the abdominal cavity in ICR mice. After 5h, cells in the abdominal cavity were then collected and stained with a primary antibody against F4/80 or IgG isotype followed by a goat anti-rat IgG-PE antibody. Cells were then analyzed by flow cytometry. Data were reported as the percent of macrophages undergoing phagocytosis, calculated by CFSE/PE double positive cells divided by all PE positive cells. Values are shown as mean ± SEM (p > 0.05, n=3). B. sCAR-PPAb enhanced the phagocytosis of K562/ADR by macrophages. Data were reported as the percent of macrophages undergoing phagocytosis, calculated by CFSE/PE double positive cells divided by all PE positive cells. Values are shown as mean ± SEM (p

    Article Snippet: Cells Human chronic myeloid leukemia cell line K562 was purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin solution, and 1% L-Glutamine.

    Techniques: In Vivo, Staining, Injection, Mouse Assay, Flow Cytometry, Cytometry

    Apoptosis is produced by activation of P73. A) Impairment of the expression of the anti-apoptotic proteins BCL-2 and XIAP is shown. B ) The decreased expression of the anti-apoptotic proteins was caspase independent, as shown by the use of Z-VAD-FMK pancaspase inhibitor. Samples were treated with 2 pulses of 40 μM of pan-caspase inhibitor, Z-VAD-FMK (R D, Systems), to 12 and 15 h of curcumin treatment. K562 cells were harvested after 18 h, lysed and total protein extracts were obtained for western blot analysis, using specific antibodies against of Caspase-3, PARP, BCL-2 and XIAP. C ) The diminution of the expression of the antiapoptotic proteins was consistent with decreases in the IκBα and D ) activation and nuclear translocation of the P73 protein, as consequence of the E) diminished expression of the P73 promoter repressor protein C/EBPα; M = medium, D = DMSO.

    Journal: PLoS ONE

    Article Title: A Subpopulation of the K562 Cells Are Killed by Curcumin Treatment after G2/M Arrest and Mitotic Catastrophe

    doi: 10.1371/journal.pone.0165971

    Figure Lengend Snippet: Apoptosis is produced by activation of P73. A) Impairment of the expression of the anti-apoptotic proteins BCL-2 and XIAP is shown. B ) The decreased expression of the anti-apoptotic proteins was caspase independent, as shown by the use of Z-VAD-FMK pancaspase inhibitor. Samples were treated with 2 pulses of 40 μM of pan-caspase inhibitor, Z-VAD-FMK (R D, Systems), to 12 and 15 h of curcumin treatment. K562 cells were harvested after 18 h, lysed and total protein extracts were obtained for western blot analysis, using specific antibodies against of Caspase-3, PARP, BCL-2 and XIAP. C ) The diminution of the expression of the antiapoptotic proteins was consistent with decreases in the IκBα and D ) activation and nuclear translocation of the P73 protein, as consequence of the E) diminished expression of the P73 promoter repressor protein C/EBPα; M = medium, D = DMSO.

    Article Snippet: Cell culture The chronic myelogenous leukemia K562 cell line was obtained from the American Type Culture Collection (ATCC® CCL-243™ ).

    Techniques: Produced, Activation Assay, Expressing, Western Blot, Translocation Assay

    Curcumin induces apoptotic cell death in mitotic K562 cells. A) A fraction of the K562 cells arrested with 20 μM of curcumin underwent caspase-dependent apoptosis as revealed by the presence of active caspase-3 in the K562 cells positive for the mitosis p-H3S10 marker and FACS analysis. B) The relative numbers of p-H3S10 positive cells with caspase-3 activity are presented as a histogram. C) Representative images of the K562 curcumin-treated cells positive for caspase-3 activity using confocal microscopy. D) Processed caspases-8, -9 and -3 were detected by western blot analysis and E) pro-caspases quantification. Activity was confirmed by cleavage of the caspase-3 substrate PARP, and its protein fragment of 89 kDa was detected. As positive controls for p-H3S10 and caspase activities, K562 cells were exposed for 24h to 100 nM Nocodazole (Noc) or 1 μM Staurosporine (Staur), respectively; M = medium, D = DMSO.

    Journal: PLoS ONE

    Article Title: A Subpopulation of the K562 Cells Are Killed by Curcumin Treatment after G2/M Arrest and Mitotic Catastrophe

    doi: 10.1371/journal.pone.0165971

    Figure Lengend Snippet: Curcumin induces apoptotic cell death in mitotic K562 cells. A) A fraction of the K562 cells arrested with 20 μM of curcumin underwent caspase-dependent apoptosis as revealed by the presence of active caspase-3 in the K562 cells positive for the mitosis p-H3S10 marker and FACS analysis. B) The relative numbers of p-H3S10 positive cells with caspase-3 activity are presented as a histogram. C) Representative images of the K562 curcumin-treated cells positive for caspase-3 activity using confocal microscopy. D) Processed caspases-8, -9 and -3 were detected by western blot analysis and E) pro-caspases quantification. Activity was confirmed by cleavage of the caspase-3 substrate PARP, and its protein fragment of 89 kDa was detected. As positive controls for p-H3S10 and caspase activities, K562 cells were exposed for 24h to 100 nM Nocodazole (Noc) or 1 μM Staurosporine (Staur), respectively; M = medium, D = DMSO.

    Article Snippet: Cell culture The chronic myelogenous leukemia K562 cell line was obtained from the American Type Culture Collection (ATCC® CCL-243™ ).

    Techniques: Marker, FACS, Activity Assay, Confocal Microscopy, Western Blot

    Curcumin induces DNA fragmentation and cell death in K562 cells. A) A fraction of the arrested K562 cells treated with 20 μM of curcumin showed DNA damage as indicated by the presence of TUNEL-positive cells and FACS analysis. B) The percentage of TUNEL-positive cells is represented by graphic bars, and C) production of a typical apoptotic DNA ladder is shown. D) Cell death was confirmed by death assays; FACS analysis, and E) the results are also shown as graphic bars. As positive controls of DNA fragmentation, we used cells treated with 100 nM nocodazole for 24 h. As a positive control of cell death, K562 cells were exposed to UV (40 mJ/cm 2 ) for 2 min in a cross-linker GS Gene linker-UV chamber (Bio-Rad, Hercules CA, USA) and were recovered 24 h post-irradiation.

    Journal: PLoS ONE

    Article Title: A Subpopulation of the K562 Cells Are Killed by Curcumin Treatment after G2/M Arrest and Mitotic Catastrophe

    doi: 10.1371/journal.pone.0165971

    Figure Lengend Snippet: Curcumin induces DNA fragmentation and cell death in K562 cells. A) A fraction of the arrested K562 cells treated with 20 μM of curcumin showed DNA damage as indicated by the presence of TUNEL-positive cells and FACS analysis. B) The percentage of TUNEL-positive cells is represented by graphic bars, and C) production of a typical apoptotic DNA ladder is shown. D) Cell death was confirmed by death assays; FACS analysis, and E) the results are also shown as graphic bars. As positive controls of DNA fragmentation, we used cells treated with 100 nM nocodazole for 24 h. As a positive control of cell death, K562 cells were exposed to UV (40 mJ/cm 2 ) for 2 min in a cross-linker GS Gene linker-UV chamber (Bio-Rad, Hercules CA, USA) and were recovered 24 h post-irradiation.

    Article Snippet: Cell culture The chronic myelogenous leukemia K562 cell line was obtained from the American Type Culture Collection (ATCC® CCL-243™ ).

    Techniques: TUNEL Assay, FACS, Positive Control, Irradiation

    Schematic representation of the mitotic catastrophe and P73-dependent apoptosis mechanisms triggered by curcumin treatment in the K562 cells.

    Journal: PLoS ONE

    Article Title: A Subpopulation of the K562 Cells Are Killed by Curcumin Treatment after G2/M Arrest and Mitotic Catastrophe

    doi: 10.1371/journal.pone.0165971

    Figure Lengend Snippet: Schematic representation of the mitotic catastrophe and P73-dependent apoptosis mechanisms triggered by curcumin treatment in the K562 cells.

    Article Snippet: Cell culture The chronic myelogenous leukemia K562 cell line was obtained from the American Type Culture Collection (ATCC® CCL-243™ ).

    Techniques:

    Mitotic spindles in K562 cells were altered by curcumin. A ) Expression levels of phosphorylsted-histone 3 in serine 10 (p-H3S10) were increased in K562 cells after curcumin treatment, as determined by western blot and B ) FACS analysis. C ) The relative numbers of p-H3S10-positive cells are presented as a histogram. K562 cells were stained with DAPI or immunostained using an antibody against α-tubulin (green fluorescence), and the cells with defects in the mitotic spindles were counted. D ) The percentage of cells with monopolar (white bars), bipolar (black bars) and multipolar (gray bars) nuclei were determined in K562 cells. E ) Representative fluorescence images of K562 cells bearing mitotic spindle alterations after 12 h, 18 h and 24 h of curcumin treatment are shown. F ) Images of the most representative mitotic spindle alterations are shown. Actin was used as loading control.

    Journal: PLoS ONE

    Article Title: A Subpopulation of the K562 Cells Are Killed by Curcumin Treatment after G2/M Arrest and Mitotic Catastrophe

    doi: 10.1371/journal.pone.0165971

    Figure Lengend Snippet: Mitotic spindles in K562 cells were altered by curcumin. A ) Expression levels of phosphorylsted-histone 3 in serine 10 (p-H3S10) were increased in K562 cells after curcumin treatment, as determined by western blot and B ) FACS analysis. C ) The relative numbers of p-H3S10-positive cells are presented as a histogram. K562 cells were stained with DAPI or immunostained using an antibody against α-tubulin (green fluorescence), and the cells with defects in the mitotic spindles were counted. D ) The percentage of cells with monopolar (white bars), bipolar (black bars) and multipolar (gray bars) nuclei were determined in K562 cells. E ) Representative fluorescence images of K562 cells bearing mitotic spindle alterations after 12 h, 18 h and 24 h of curcumin treatment are shown. F ) Images of the most representative mitotic spindle alterations are shown. Actin was used as loading control.

    Article Snippet: Cell culture The chronic myelogenous leukemia K562 cell line was obtained from the American Type Culture Collection (ATCC® CCL-243™ ).

    Techniques: Expressing, Western Blot, FACS, Staining, Fluorescence

    K562 cells treated with curcumin were arrested in G2/M2 phase. A) A subpopulation of the curcumin-treated K562 cells was arrested in G2/M phase, as determined by FACS analysis. Cells were stained with DAPI or immunostained with rhodamine-phalloidin (red fluorescence) to visualize the nuclei and actin fibers, respectively. B ) Subsequently, they were analyzed by immunofluorescence microscopy, and the percentage of giant or multinucleated cells was determined. C ) The amplification of these cells clearly showed the condensed chromatin and the actin cytoskeleton changes due to early mitotic events. Condensed nuclei were considered an indirect measure of mitotic cells. As a positive control for the enriched G2/M population, K562 cells were treated for 24 h with 100 nM nocodazole (Noc). IN = interphase nucleus; mN = multinucleated cell.

    Journal: PLoS ONE

    Article Title: A Subpopulation of the K562 Cells Are Killed by Curcumin Treatment after G2/M Arrest and Mitotic Catastrophe

    doi: 10.1371/journal.pone.0165971

    Figure Lengend Snippet: K562 cells treated with curcumin were arrested in G2/M2 phase. A) A subpopulation of the curcumin-treated K562 cells was arrested in G2/M phase, as determined by FACS analysis. Cells were stained with DAPI or immunostained with rhodamine-phalloidin (red fluorescence) to visualize the nuclei and actin fibers, respectively. B ) Subsequently, they were analyzed by immunofluorescence microscopy, and the percentage of giant or multinucleated cells was determined. C ) The amplification of these cells clearly showed the condensed chromatin and the actin cytoskeleton changes due to early mitotic events. Condensed nuclei were considered an indirect measure of mitotic cells. As a positive control for the enriched G2/M population, K562 cells were treated for 24 h with 100 nM nocodazole (Noc). IN = interphase nucleus; mN = multinucleated cell.

    Article Snippet: Cell culture The chronic myelogenous leukemia K562 cell line was obtained from the American Type Culture Collection (ATCC® CCL-243™ ).

    Techniques: FACS, Staining, Fluorescence, Immunofluorescence, Microscopy, Amplification, Positive Control

    Influence of the tested inhibitors – TOS-1, TOS-2 and axitinib on 2-HG production in the K562 ( A ) and U-251 cells ( B ). Data were analyzed using a one-way ANOVA with Bonferroni’s post-hoc test: ** p

    Journal: Cancers

    Article Title: The Landscape of the Anti-Kinase Activity of the IDH1 Inhibitors

    doi: 10.3390/cancers12030536

    Figure Lengend Snippet: Influence of the tested inhibitors – TOS-1, TOS-2 and axitinib on 2-HG production in the K562 ( A ) and U-251 cells ( B ). Data were analyzed using a one-way ANOVA with Bonferroni’s post-hoc test: ** p

    Article Snippet: The pancreatic cell line PANC-1 and the human chronic myelogenous leukemia cell line K562 were purchased from Sigma-Aldrich.

    Techniques:

    RM GFP and CD20 CAR T cell product composition and function A, GFP and EGFRt expression in RM T cell products transduced with GFP (R.301 GFP, green) or CD20 CAR (R.301–R.304 EGFRt, red); Mock (blue). CD8 and CD4 composition in RM GFP+ (R.301 GFP+) or EGFRt+ T cell products (R.301–R.304 EGFRt+). B, Composition of T cells in CD20 CAR products in EGFRt+ (top) and EGFRt− (bottom) CD4 and CD8 cells (R.301–R.304, n = 4). CD28+/CD95+: central memory, CD28−/CD95+: effector memory and CD28+/CD95−: naive T cells. Horizontal lines represent the mean. C, CD20 antigen expression in human (K562 and CD20-K562), and in RM (B-LCL1 and B-LCL2) cell lines. Cytolytic activity of RM mock-transduced (dashed lines) and CD20 CAR T cells (solid lines) against 51 Cr-labeled targets (K562, CD20-K562, B-LCL1 and B-LCL2). n = 3 replicates per point; representative of four recipients.

    Journal: Cancer discovery

    Article Title: Chimeric Antigen Receptor T Cell-Mediated Neurotoxicity in Non-Human Primates

    doi: 10.1158/2159-8290.CD-17-1368

    Figure Lengend Snippet: RM GFP and CD20 CAR T cell product composition and function A, GFP and EGFRt expression in RM T cell products transduced with GFP (R.301 GFP, green) or CD20 CAR (R.301–R.304 EGFRt, red); Mock (blue). CD8 and CD4 composition in RM GFP+ (R.301 GFP+) or EGFRt+ T cell products (R.301–R.304 EGFRt+). B, Composition of T cells in CD20 CAR products in EGFRt+ (top) and EGFRt− (bottom) CD4 and CD8 cells (R.301–R.304, n = 4). CD28+/CD95+: central memory, CD28−/CD95+: effector memory and CD28+/CD95−: naive T cells. Horizontal lines represent the mean. C, CD20 antigen expression in human (K562 and CD20-K562), and in RM (B-LCL1 and B-LCL2) cell lines. Cytolytic activity of RM mock-transduced (dashed lines) and CD20 CAR T cells (solid lines) against 51 Cr-labeled targets (K562, CD20-K562, B-LCL1 and B-LCL2). n = 3 replicates per point; representative of four recipients.

    Article Snippet: The human K562 chronic myelogenous leukemia (CML) cell line was obtained from the European Collection of Cell Cultures through Sigma-Aldrich in 2012.

    Techniques: Expressing, Transduction, Activity Assay, Labeling

    K1K2 polypeptide inhibits rIFN-γ-induced HLA-1 surface expression. (A) HLA-1 expression by K562 cells at 72 h after treatment with rIFN-γ alone at 24 nM or with rIFN-γ pretreated with increasing levels of K1K2 or K1K2K3. (B) HLA-1 surface expression on K562 cells at 72 h following either no treatment or addition of rIFN-γ (pretreated with or without adhesin domains [molar ratio, 1:100]). To assess the impact of additional domains, K1K2 at 2.4 μM was pretreated with K3 for 6 h at 4°C at equal molar ratio before addition to rIFN-γ. (C) Panels i to vii show FACS histogram representations of HLA-1 expression at 72 h after rIFN-γ treatment. The interferon was pretreated with K1K2 for 6 h or 24 h or with K1K2K3, K1, K2, K3 for 24 h. The shaded areas represent the fluorescence levels of HLA-1 expression induced by IFN-γ after 3 days of culture. The broken lines in panels ii to vii represent background values, while the bold black lines represent IFN-γ pretreated with different adhesin polypeptides before the addition to culture medium. The results are representative of those from three experiments. Means ± SEM are shown. *, P

    Journal: Infection and Immunity

    Article Title: The K1K2 Region of Lys-Gingipain of Porphyromonas gingivalis Blocks Induction of HLA Expression by Gamma Interferon

    doi: 10.1128/IAI.00528-12

    Figure Lengend Snippet: K1K2 polypeptide inhibits rIFN-γ-induced HLA-1 surface expression. (A) HLA-1 expression by K562 cells at 72 h after treatment with rIFN-γ alone at 24 nM or with rIFN-γ pretreated with increasing levels of K1K2 or K1K2K3. (B) HLA-1 surface expression on K562 cells at 72 h following either no treatment or addition of rIFN-γ (pretreated with or without adhesin domains [molar ratio, 1:100]). To assess the impact of additional domains, K1K2 at 2.4 μM was pretreated with K3 for 6 h at 4°C at equal molar ratio before addition to rIFN-γ. (C) Panels i to vii show FACS histogram representations of HLA-1 expression at 72 h after rIFN-γ treatment. The interferon was pretreated with K1K2 for 6 h or 24 h or with K1K2K3, K1, K2, K3 for 24 h. The shaded areas represent the fluorescence levels of HLA-1 expression induced by IFN-γ after 3 days of culture. The broken lines in panels ii to vii represent background values, while the bold black lines represent IFN-γ pretreated with different adhesin polypeptides before the addition to culture medium. The results are representative of those from three experiments. Means ± SEM are shown. *, P

    Article Snippet: K562 (human myelogenous leukemia cell line) cells ( ) were purchased from Sigma-Aldrich, Castle Hill, New South Wales, Australia.

    Techniques: Expressing, FACS, Fluorescence

    Effects of anti-K2 Ab on the binding of rIFN-γ with K1K2. K1K2 was pretreated with anti-K2 Ab or control Ab overnight and then incubated with rIFN-γ for 24 h. In addition, 5AI Ab was preincubated in the presence or absence of K1K2 before addition to rIFN-γ. After incubation, all mixtures were added to the K562 cells, and the HLA-1 expression at 72 h was determined. Results are representative of three separate experiments. Means ± SEM are shown.

    Journal: Infection and Immunity

    Article Title: The K1K2 Region of Lys-Gingipain of Porphyromonas gingivalis Blocks Induction of HLA Expression by Gamma Interferon

    doi: 10.1128/IAI.00528-12

    Figure Lengend Snippet: Effects of anti-K2 Ab on the binding of rIFN-γ with K1K2. K1K2 was pretreated with anti-K2 Ab or control Ab overnight and then incubated with rIFN-γ for 24 h. In addition, 5AI Ab was preincubated in the presence or absence of K1K2 before addition to rIFN-γ. After incubation, all mixtures were added to the K562 cells, and the HLA-1 expression at 72 h was determined. Results are representative of three separate experiments. Means ± SEM are shown.

    Article Snippet: K562 (human myelogenous leukemia cell line) cells ( ) were purchased from Sigma-Aldrich, Castle Hill, New South Wales, Australia.

    Techniques: Binding Assay, Incubation, Expressing

    K1K2 polypeptide modulates expression of IFN-γR1 and IFN-γR2 in K562 cells. (A and B) K562 cells were grown and incubated with rIFN-γ (A) or IFN-γ peptide 1-14 (B) pretreated overnight with recombinant adhesin domains as shown, and the mixtures were further incubated for 72 h at 37°C. FACS histograms show the cell staining for surface expression of IFN-γR1 and IFN-γR2 on K562 cells. The results are representative of those from three experiments. Means ± SEM are shown. *, P

    Journal: Infection and Immunity

    Article Title: The K1K2 Region of Lys-Gingipain of Porphyromonas gingivalis Blocks Induction of HLA Expression by Gamma Interferon

    doi: 10.1128/IAI.00528-12

    Figure Lengend Snippet: K1K2 polypeptide modulates expression of IFN-γR1 and IFN-γR2 in K562 cells. (A and B) K562 cells were grown and incubated with rIFN-γ (A) or IFN-γ peptide 1-14 (B) pretreated overnight with recombinant adhesin domains as shown, and the mixtures were further incubated for 72 h at 37°C. FACS histograms show the cell staining for surface expression of IFN-γR1 and IFN-γR2 on K562 cells. The results are representative of those from three experiments. Means ± SEM are shown. *, P

    Article Snippet: K562 (human myelogenous leukemia cell line) cells ( ) were purchased from Sigma-Aldrich, Castle Hill, New South Wales, Australia.

    Techniques: Expressing, Incubation, Recombinant, FACS, Staining

    K1K2 polypeptide binds to and inhibits IFN-γ 1-14-induced HLA-1 surface expression. (A) ELISA plates were coated with K1K2-, K1K2K3-, K1-, K2-, K3-, and TLCK-treated Kgp proteins (1 μg/well in PBS) and incubated overnight a 4°C. IFN-γ 1-14 peptide was added to the plates in various concentrations. Thereafter, anti-IFN-γ MAb was added, followed by alkaline phosphatase-conjugated rabbit anti-mouse IgG. (B) The percentage of HLA-1 expression in K562 cells at 72 h was shown by flow cytometry after treatment with IFN-γ 1-14 peptide at different concentrations or with K1K2/K1K2K3 at 2 μM each pretreated with increasing levels of IFN-γ 1-14 peptide for 24 h. Results are representative of three separate experiments. Means and SE are shown. *, P

    Journal: Infection and Immunity

    Article Title: The K1K2 Region of Lys-Gingipain of Porphyromonas gingivalis Blocks Induction of HLA Expression by Gamma Interferon

    doi: 10.1128/IAI.00528-12

    Figure Lengend Snippet: K1K2 polypeptide binds to and inhibits IFN-γ 1-14-induced HLA-1 surface expression. (A) ELISA plates were coated with K1K2-, K1K2K3-, K1-, K2-, K3-, and TLCK-treated Kgp proteins (1 μg/well in PBS) and incubated overnight a 4°C. IFN-γ 1-14 peptide was added to the plates in various concentrations. Thereafter, anti-IFN-γ MAb was added, followed by alkaline phosphatase-conjugated rabbit anti-mouse IgG. (B) The percentage of HLA-1 expression in K562 cells at 72 h was shown by flow cytometry after treatment with IFN-γ 1-14 peptide at different concentrations or with K1K2/K1K2K3 at 2 μM each pretreated with increasing levels of IFN-γ 1-14 peptide for 24 h. Results are representative of three separate experiments. Means and SE are shown. *, P

    Article Snippet: K562 (human myelogenous leukemia cell line) cells ( ) were purchased from Sigma-Aldrich, Castle Hill, New South Wales, Australia.

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry, Cytometry

    K1K2 polypeptide downregulates native IFN-γ (nIFN-γ)-induced HLA-1 expression. K562 cells were grown and incubated with nIFN-γ pretreated overnight with K1K2, K1K2K3, K2, or TLCK-treated Kgp (KgpTL) in a molar ratio of 1:100, and the mixtures were further incubated for 72 h at 37°C. FACS histograms show the cell staining for surface expression of HLA-1 on K562 cells. The results are representative of those from three experiments.

    Journal: Infection and Immunity

    Article Title: The K1K2 Region of Lys-Gingipain of Porphyromonas gingivalis Blocks Induction of HLA Expression by Gamma Interferon

    doi: 10.1128/IAI.00528-12

    Figure Lengend Snippet: K1K2 polypeptide downregulates native IFN-γ (nIFN-γ)-induced HLA-1 expression. K562 cells were grown and incubated with nIFN-γ pretreated overnight with K1K2, K1K2K3, K2, or TLCK-treated Kgp (KgpTL) in a molar ratio of 1:100, and the mixtures were further incubated for 72 h at 37°C. FACS histograms show the cell staining for surface expression of HLA-1 on K562 cells. The results are representative of those from three experiments.

    Article Snippet: K562 (human myelogenous leukemia cell line) cells ( ) were purchased from Sigma-Aldrich, Castle Hill, New South Wales, Australia.

    Techniques: Expressing, Incubation, FACS, Staining

    Effects of different salts on the binding of rIFN-γ with K1K2. rIFN-γ was pretreated with or without K1K2 (A to C) or K1K2K3 (A) in the presence or absence of different salts, including NaCl, KCl, and KBr, for 24 h at 4°C. After incubation, all mixtures were added to the K562 cells and left for 72 h at 37°C. Both NaCl and KCl can eliminate the K1K2 inhibition of rIFN-γ activities in a dose-dependent manner.

    Journal: Infection and Immunity

    Article Title: The K1K2 Region of Lys-Gingipain of Porphyromonas gingivalis Blocks Induction of HLA Expression by Gamma Interferon

    doi: 10.1128/IAI.00528-12

    Figure Lengend Snippet: Effects of different salts on the binding of rIFN-γ with K1K2. rIFN-γ was pretreated with or without K1K2 (A to C) or K1K2K3 (A) in the presence or absence of different salts, including NaCl, KCl, and KBr, for 24 h at 4°C. After incubation, all mixtures were added to the K562 cells and left for 72 h at 37°C. Both NaCl and KCl can eliminate the K1K2 inhibition of rIFN-γ activities in a dose-dependent manner.

    Article Snippet: K562 (human myelogenous leukemia cell line) cells ( ) were purchased from Sigma-Aldrich, Castle Hill, New South Wales, Australia.

    Techniques: Binding Assay, Incubation, Inhibition

    Single treatment of DOR and dual treatments of BMP inhibitors and IM inhibit cell proliferation of CML CD34+ cells. a , b CellTrace™ Violet (CTV) proliferation analysis of normal and CP-CML CD34 + cells treated with IM, BMP inhibitors, the combination (IM = 1μM, LDN = 1 μM, DOR = 2.5 μM, n = 3), in absence or presence of BMP4 at 72 h. i Results for normal CD34 + cells display that single and dual treatment of IM + DOR inhibit cell proliferation, and additional BMP4 treatment ( b ) results in more cell divisions compared to standard culture conditions without BMP4. a-ii Proliferation analysis of CP-CML samples indicates that LDN in combination with IM synergistically inhibits proliferation compared to single treatment. DOR alone and in combination with IM displays the biggest fold change compared to NDC. Additional treatment with BMP4 ( b-ii ) did not reveal differences in proliferation progression compared to standard culture conditions. c IM and BMP inhibitors reduce CD34 + cell numbers in CP-CML samples in absence or presence of BMP4. Results are displayed as median range of CD34 + cell numbers of normal and CP-CML CD34 + cells treated with IM, BMP inhibitors and the combination (IM = 1 µM, LDN = 1 µM, DOR = 2.5 μM, n = 3) in absence or presence of BMP4. d-i CFC assay results of normal CD34 + cells treated with IM, BMP inhibitors and the combination of (IM = 1 µM, LDN = 1 µM, DOR = 2.5 μM) do not reveal any significant difference compared to NDC ( n = 3). ii Total number of colonies of CP-CML CD34 + cells display a significant difference across all treatments compared to NDC and a synergistic reduction when IM and DOR were used in combination ( n = 3). iii CFC results of K562 cells. Results also indicate a significant drop in the number of colonies with IM, DOR and LDN. Results are also reflected in the morphology of the colonies with DOR and LDN treatment reducing the size of colonies ( n = 3). Data are expressed as mean ± standard deviation and were compared using ANOVA ( c ) and the unpaired Student’s t -test ( a , b , d ), *** p

    Journal: Cell Death & Disease

    Article Title: Chronic myeloid leukaemia cells require the bone morphogenic protein pathway for cell cycle progression and self-renewal

    doi: 10.1038/s41419-018-0905-2

    Figure Lengend Snippet: Single treatment of DOR and dual treatments of BMP inhibitors and IM inhibit cell proliferation of CML CD34+ cells. a , b CellTrace™ Violet (CTV) proliferation analysis of normal and CP-CML CD34 + cells treated with IM, BMP inhibitors, the combination (IM = 1μM, LDN = 1 μM, DOR = 2.5 μM, n = 3), in absence or presence of BMP4 at 72 h. i Results for normal CD34 + cells display that single and dual treatment of IM + DOR inhibit cell proliferation, and additional BMP4 treatment ( b ) results in more cell divisions compared to standard culture conditions without BMP4. a-ii Proliferation analysis of CP-CML samples indicates that LDN in combination with IM synergistically inhibits proliferation compared to single treatment. DOR alone and in combination with IM displays the biggest fold change compared to NDC. Additional treatment with BMP4 ( b-ii ) did not reveal differences in proliferation progression compared to standard culture conditions. c IM and BMP inhibitors reduce CD34 + cell numbers in CP-CML samples in absence or presence of BMP4. Results are displayed as median range of CD34 + cell numbers of normal and CP-CML CD34 + cells treated with IM, BMP inhibitors and the combination (IM = 1 µM, LDN = 1 µM, DOR = 2.5 μM, n = 3) in absence or presence of BMP4. d-i CFC assay results of normal CD34 + cells treated with IM, BMP inhibitors and the combination of (IM = 1 µM, LDN = 1 µM, DOR = 2.5 μM) do not reveal any significant difference compared to NDC ( n = 3). ii Total number of colonies of CP-CML CD34 + cells display a significant difference across all treatments compared to NDC and a synergistic reduction when IM and DOR were used in combination ( n = 3). iii CFC results of K562 cells. Results also indicate a significant drop in the number of colonies with IM, DOR and LDN. Results are also reflected in the morphology of the colonies with DOR and LDN treatment reducing the size of colonies ( n = 3). Data are expressed as mean ± standard deviation and were compared using ANOVA ( c ) and the unpaired Student’s t -test ( a , b , d ), *** p

    Article Snippet: Half maximal inhibitory concentration (IC50) of each of the BMP pathway inhibitors and IM in K562 cell line was established using XTT sodium 3′-[1-[(phenylamino)-carbonyl]-3,4-Tetrazzolium]-bis(4-methoxy-6-nitro) benzene sulphonic acid hydrate cell proliferation assay (Sigma-Aldrich) according to manufacturer’s instructions.

    Techniques: Standard Deviation

    IM and BMP pathway inhibitors synergistically target the K562 CML cell line. a Synergy studies of the BMP pathway inhibitors, LDN-193189 (LDN) and Dorsomorphin (DOR) with imatinib mesylate (IM) were performed using CalcuSyn to quantify additive, synergistic and inhibitory effects. Tables present the fraction of cells affected (Fa) and combination index (CI) values at different combination concentrations. The bottom table describes the correlation between CI range and synergism. As shown, both LDN and DOR, in combination with IM, synergistically induce cell death in K562 cells. b Protein analysis of K562 cells treated with IM, BMP inhibitors and the combination (IM = 500 nM, LDN = 500 nM, DOR = 2.5 μM, n = 3) compared to no-drug control (NDC) using western blot hybridisation following drug treatment in the presence and absence of BMP4 stimulation. i IM and LDN combination immunoblots at 4 h (left panel) and IM and DOR combination immunoblots at 4 h (right panel). K562 cells were stimulated with 20 ng/mL BMP4 for 30 min; this was followed by drug treatment. SHP2 was used as the housekeeping protein. ii Densitometry analysis of western blots was performed using image J software. Analysis was normalised relative to the housekeeping protein expression. Both BMP inhibitors synergistically inhibit pCrKl significantly in the absence of BMP4 stimulation. Data are expressed as mean ± standard deviation and were compared using the unpaired Student’s t -test, * p

    Journal: Cell Death & Disease

    Article Title: Chronic myeloid leukaemia cells require the bone morphogenic protein pathway for cell cycle progression and self-renewal

    doi: 10.1038/s41419-018-0905-2

    Figure Lengend Snippet: IM and BMP pathway inhibitors synergistically target the K562 CML cell line. a Synergy studies of the BMP pathway inhibitors, LDN-193189 (LDN) and Dorsomorphin (DOR) with imatinib mesylate (IM) were performed using CalcuSyn to quantify additive, synergistic and inhibitory effects. Tables present the fraction of cells affected (Fa) and combination index (CI) values at different combination concentrations. The bottom table describes the correlation between CI range and synergism. As shown, both LDN and DOR, in combination with IM, synergistically induce cell death in K562 cells. b Protein analysis of K562 cells treated with IM, BMP inhibitors and the combination (IM = 500 nM, LDN = 500 nM, DOR = 2.5 μM, n = 3) compared to no-drug control (NDC) using western blot hybridisation following drug treatment in the presence and absence of BMP4 stimulation. i IM and LDN combination immunoblots at 4 h (left panel) and IM and DOR combination immunoblots at 4 h (right panel). K562 cells were stimulated with 20 ng/mL BMP4 for 30 min; this was followed by drug treatment. SHP2 was used as the housekeeping protein. ii Densitometry analysis of western blots was performed using image J software. Analysis was normalised relative to the housekeeping protein expression. Both BMP inhibitors synergistically inhibit pCrKl significantly in the absence of BMP4 stimulation. Data are expressed as mean ± standard deviation and were compared using the unpaired Student’s t -test, * p

    Article Snippet: Half maximal inhibitory concentration (IC50) of each of the BMP pathway inhibitors and IM in K562 cell line was established using XTT sodium 3′-[1-[(phenylamino)-carbonyl]-3,4-Tetrazzolium]-bis(4-methoxy-6-nitro) benzene sulphonic acid hydrate cell proliferation assay (Sigma-Aldrich) according to manufacturer’s instructions.

    Techniques: Western Blot, Hybridization, Software, Expressing, Standard Deviation

    IM and BMP inhibitors induce apoptosis in CML cells. a Annexin V/ 7AAD apoptosis analysis of K562 cells treated with IM, BMP inhibitors and the combination (IM = 500 nM, LDN = 500 nM, DOR = 2.5 μM, n = 3) at 72 h. i Inhibition of BCR-ABL and BMP pathway results in apoptosis, and a reduction in viable cells. ii Results are significantly more profound when IM is used in combination with DOR with more cells in early apoptosis ( n = 3). b Annexin V/7AAD apoptosis analysis of normal and CP-CML CD34 + cells treated with IM, BMP inhibitors and the combination (IM = 1 µM, LDN = 1 µM, DOR = 2.5 μM, n = 3) at 72 h. i, ii Results indicate that IM in combination with BMP pathway inhibitors promote modest, but significant, apoptosis in normal CD34 + cells. However, apoptosis is greatly increased in CP-CML CD34 + samples. This is reflected in the increased number of cells present in late apoptosis ( n = 3). iii, iv Inhibitor treatments with addition of BMP4 also indicate a significant increase of normal and CP-CML CD34 + cells in late apoptosis, with more apoptosis observed in the CML CD34 + samples ( n = 3)

    Journal: Cell Death & Disease

    Article Title: Chronic myeloid leukaemia cells require the bone morphogenic protein pathway for cell cycle progression and self-renewal

    doi: 10.1038/s41419-018-0905-2

    Figure Lengend Snippet: IM and BMP inhibitors induce apoptosis in CML cells. a Annexin V/ 7AAD apoptosis analysis of K562 cells treated with IM, BMP inhibitors and the combination (IM = 500 nM, LDN = 500 nM, DOR = 2.5 μM, n = 3) at 72 h. i Inhibition of BCR-ABL and BMP pathway results in apoptosis, and a reduction in viable cells. ii Results are significantly more profound when IM is used in combination with DOR with more cells in early apoptosis ( n = 3). b Annexin V/7AAD apoptosis analysis of normal and CP-CML CD34 + cells treated with IM, BMP inhibitors and the combination (IM = 1 µM, LDN = 1 µM, DOR = 2.5 μM, n = 3) at 72 h. i, ii Results indicate that IM in combination with BMP pathway inhibitors promote modest, but significant, apoptosis in normal CD34 + cells. However, apoptosis is greatly increased in CP-CML CD34 + samples. This is reflected in the increased number of cells present in late apoptosis ( n = 3). iii, iv Inhibitor treatments with addition of BMP4 also indicate a significant increase of normal and CP-CML CD34 + cells in late apoptosis, with more apoptosis observed in the CML CD34 + samples ( n = 3)

    Article Snippet: Half maximal inhibitory concentration (IC50) of each of the BMP pathway inhibitors and IM in K562 cell line was established using XTT sodium 3′-[1-[(phenylamino)-carbonyl]-3,4-Tetrazzolium]-bis(4-methoxy-6-nitro) benzene sulphonic acid hydrate cell proliferation assay (Sigma-Aldrich) according to manufacturer’s instructions.

    Techniques: Inhibition

    Modulation of degranulation (CD107a) and perforin secretion in NK cell subsets. Flow cytometric analysis for CD107a surface marker showed a downregulation of CD107a expression on PB-NK and PE-NK cells in terms of percentage of positive cells (a) and mean intensity of fluorescence (MFI) (b) that correlated with a significant decrease of the percentage of perforin + NK cells, both in CD56 dim and CD56 bright subsets in iPE and ptPE (c, d) after exposure to target K562 cells. Representative dot plots of perforin expression in healthy donors and patients with inflammatory, primary, and metastatic tumor PE are shown, respectively (e). Data are shown as mean ± SEM of 34 samples; ∗ p

    Journal: Journal of Immunology Research

    Article Title: Natural Killer Cells from Malignant Pleural Effusion Are Endowed with a Decidual-Like Proangiogenic Polarization

    doi: 10.1155/2018/2438598

    Figure Lengend Snippet: Modulation of degranulation (CD107a) and perforin secretion in NK cell subsets. Flow cytometric analysis for CD107a surface marker showed a downregulation of CD107a expression on PB-NK and PE-NK cells in terms of percentage of positive cells (a) and mean intensity of fluorescence (MFI) (b) that correlated with a significant decrease of the percentage of perforin + NK cells, both in CD56 dim and CD56 bright subsets in iPE and ptPE (c, d) after exposure to target K562 cells. Representative dot plots of perforin expression in healthy donors and patients with inflammatory, primary, and metastatic tumor PE are shown, respectively (e). Data are shown as mean ± SEM of 34 samples; ∗ p

    Article Snippet: Detection of NK Cell Degranulation Capacity The NK cell degranulation activity assay was performed on total mononuclear cells from PB and PE after in vitro 4 h incubation with the K562 tumor cell line human target (chronic myelogenous leukemia, ECACC, Sigma-Aldrich) in the presence of conjugated anti-CD107a mAb (Miltenyi Biotec) and monensin (2 mM, BD) at a NK: target cell ratio of 1 : 1 [ ].

    Techniques: Flow Cytometry, Marker, Expressing, Fluorescence

    Induction of NK cell anergy by TGF β or PE fluids in the presence of IL-2. IL-2, TGF β , or autologous PE fluid treatment of NK cells, both from autologous or healthy control-derived NK cells, was performed every 2 days starting from day 0 (D0) for three days as shown by the timeline (a). At day 3 (D3) following coculture with target K562 cells, CD107a modulation was detected by flow cytometry. After IL-2 exposure, PB-NK and PE-NK from patients with PE showed an enhanced cytotoxic potential associated with an increased expression of CD107a. The addition of TGF β or inflammatory or metastatic PE supernatants strongly inhibited the IL-2-induced stimulation, inducing a downregulation of CD107a surface marker (b–d). Data are shown as mean ± SEM of 38 samples; ∗ p

    Journal: Journal of Immunology Research

    Article Title: Natural Killer Cells from Malignant Pleural Effusion Are Endowed with a Decidual-Like Proangiogenic Polarization

    doi: 10.1155/2018/2438598

    Figure Lengend Snippet: Induction of NK cell anergy by TGF β or PE fluids in the presence of IL-2. IL-2, TGF β , or autologous PE fluid treatment of NK cells, both from autologous or healthy control-derived NK cells, was performed every 2 days starting from day 0 (D0) for three days as shown by the timeline (a). At day 3 (D3) following coculture with target K562 cells, CD107a modulation was detected by flow cytometry. After IL-2 exposure, PB-NK and PE-NK from patients with PE showed an enhanced cytotoxic potential associated with an increased expression of CD107a. The addition of TGF β or inflammatory or metastatic PE supernatants strongly inhibited the IL-2-induced stimulation, inducing a downregulation of CD107a surface marker (b–d). Data are shown as mean ± SEM of 38 samples; ∗ p

    Article Snippet: Detection of NK Cell Degranulation Capacity The NK cell degranulation activity assay was performed on total mononuclear cells from PB and PE after in vitro 4 h incubation with the K562 tumor cell line human target (chronic myelogenous leukemia, ECACC, Sigma-Aldrich) in the presence of conjugated anti-CD107a mAb (Miltenyi Biotec) and monensin (2 mM, BD) at a NK: target cell ratio of 1 : 1 [ ].

    Techniques: Derivative Assay, Flow Cytometry, Cytometry, Expressing, Marker

    Change in SLC22A1 expression in K562 cells treated with 0.1 μM to 10 μM imatinib compared to the untreated control. Statistically significant differences (p

    Journal: Medical Science Monitor Basic Research

    Article Title: Imatinib Affects the Expression of SLC22A1 in a Non-Linear Concentration-Dependent Manner Within 24 Hours

    doi: 10.12659/MSMBR.909124

    Figure Lengend Snippet: Change in SLC22A1 expression in K562 cells treated with 0.1 μM to 10 μM imatinib compared to the untreated control. Statistically significant differences (p

    Article Snippet: Cell culture The human CML cell line, K562, was cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal bovine serum (Sigma-Aldrich), 1% penicillin-streptomycin (Sigma-Aldrich), and 5 μg/ml Plasmocin (InvivoGen) at 37°C under a 5% CO2 atmosphere.

    Techniques: Expressing

    Aberrant expression of miR-181a in CML. Reverse transcription-quantitative polymerase chain reaction analysis revealed lower expression levels of miR-181a in 1 patient with CML and in CML K562 cells, compared with the healthy volunteer control group (P

    Journal: Oncology Letters

    Article Title: MicroRNA-181a enhances the chemotherapeutic sensitivity of chronic myeloid leukemia to imatinib

    doi: 10.3892/ol.2015.3663

    Figure Lengend Snippet: Aberrant expression of miR-181a in CML. Reverse transcription-quantitative polymerase chain reaction analysis revealed lower expression levels of miR-181a in 1 patient with CML and in CML K562 cells, compared with the healthy volunteer control group (P

    Article Snippet: Induction and determination of K562 cell differentiation The K562 cells were seeded at a density of 5×105 cells/ml in DMEM, supplemented with 50 µM hemin (Sigma-Aldrich, St. Louis, USA) to induce erythroid differentiation or 25 µM TPA (Sigma-Aldrich) for megakaryocytic differentiation.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Overexpression of miR-181a promotes the megakaryocytic differentiation of chronic myeloid leukemia K562 cells. The overexpression of miR-181a induced the expression of (A) CD41 and (B) CD61 in the K562-miR-181a cells (*P

    Journal: Oncology Letters

    Article Title: MicroRNA-181a enhances the chemotherapeutic sensitivity of chronic myeloid leukemia to imatinib

    doi: 10.3892/ol.2015.3663

    Figure Lengend Snippet: Overexpression of miR-181a promotes the megakaryocytic differentiation of chronic myeloid leukemia K562 cells. The overexpression of miR-181a induced the expression of (A) CD41 and (B) CD61 in the K562-miR-181a cells (*P

    Article Snippet: Induction and determination of K562 cell differentiation The K562 cells were seeded at a density of 5×105 cells/ml in DMEM, supplemented with 50 µM hemin (Sigma-Aldrich, St. Louis, USA) to induce erythroid differentiation or 25 µM TPA (Sigma-Aldrich) for megakaryocytic differentiation.

    Techniques: Over Expression, Expressing

    Overexpression of miR-181a in chronic myeloid leukemia K562 cells promotes the apoptosis of K562-miR-181a cells induced by the chemotherapeutic agent imatinib. (A) The apoptotic K562-miR-181a and K562-EGFP cells were stained with PE-Annexin V and 7-AAD, and detected by flow cytometry. (B) Graphical representation of the percentage of K562 cells undergoing apoptosis at different stages of the process (early, mid and late apoptosis), compared with the percentage of healthy cells. K562-EGFP cells were used as the control. miR-181a, microRNA-181a; 7-AAD, 7-aminoactinomycin D; PE, phycoerythrin; EGFP, enhanced green fluorescent protein.

    Journal: Oncology Letters

    Article Title: MicroRNA-181a enhances the chemotherapeutic sensitivity of chronic myeloid leukemia to imatinib

    doi: 10.3892/ol.2015.3663

    Figure Lengend Snippet: Overexpression of miR-181a in chronic myeloid leukemia K562 cells promotes the apoptosis of K562-miR-181a cells induced by the chemotherapeutic agent imatinib. (A) The apoptotic K562-miR-181a and K562-EGFP cells were stained with PE-Annexin V and 7-AAD, and detected by flow cytometry. (B) Graphical representation of the percentage of K562 cells undergoing apoptosis at different stages of the process (early, mid and late apoptosis), compared with the percentage of healthy cells. K562-EGFP cells were used as the control. miR-181a, microRNA-181a; 7-AAD, 7-aminoactinomycin D; PE, phycoerythrin; EGFP, enhanced green fluorescent protein.

    Article Snippet: Induction and determination of K562 cell differentiation The K562 cells were seeded at a density of 5×105 cells/ml in DMEM, supplemented with 50 µM hemin (Sigma-Aldrich, St. Louis, USA) to induce erythroid differentiation or 25 µM TPA (Sigma-Aldrich) for megakaryocytic differentiation.

    Techniques: Over Expression, Staining, Flow Cytometry, Cytometry

    Overexpression of miR-181a in chronic myeloid leukemia K562 cells inhibits the proliferation of these cells by repressing the transition from the G 1 to S phase of the cell cycle. (A) miR-181a inhibited the growth of the K562 cells overexpressing miR-181a compared with the control K562-EGFP cells. (B) miR-181a prevented the K562 cells from undergoing the G 1 /S phase transition of the cell cycle. Compared with the control cells, a larger number of K562-miR-181a cells were arrested at the G 1 phase of the cell cycle, whereas the opposite was observed for the number of cells arrested at the S phase of the cell cycle. miR-181a, microRNA-181a; EGFP, enhanced green fluorescent protein.

    Journal: Oncology Letters

    Article Title: MicroRNA-181a enhances the chemotherapeutic sensitivity of chronic myeloid leukemia to imatinib

    doi: 10.3892/ol.2015.3663

    Figure Lengend Snippet: Overexpression of miR-181a in chronic myeloid leukemia K562 cells inhibits the proliferation of these cells by repressing the transition from the G 1 to S phase of the cell cycle. (A) miR-181a inhibited the growth of the K562 cells overexpressing miR-181a compared with the control K562-EGFP cells. (B) miR-181a prevented the K562 cells from undergoing the G 1 /S phase transition of the cell cycle. Compared with the control cells, a larger number of K562-miR-181a cells were arrested at the G 1 phase of the cell cycle, whereas the opposite was observed for the number of cells arrested at the S phase of the cell cycle. miR-181a, microRNA-181a; EGFP, enhanced green fluorescent protein.

    Article Snippet: Induction and determination of K562 cell differentiation The K562 cells were seeded at a density of 5×105 cells/ml in DMEM, supplemented with 50 µM hemin (Sigma-Aldrich, St. Louis, USA) to induce erythroid differentiation or 25 µM TPA (Sigma-Aldrich) for megakaryocytic differentiation.

    Techniques: Over Expression, Sublimation

    Overexpression of miR-181a promotes the erythroid differentiation of chronic myeloid leukemia K562 cells. The overexpression of miR-181a induced the expression of (A) CD235a and (B) γ-globin in the K562-miR-181a cells (*P

    Journal: Oncology Letters

    Article Title: MicroRNA-181a enhances the chemotherapeutic sensitivity of chronic myeloid leukemia to imatinib

    doi: 10.3892/ol.2015.3663

    Figure Lengend Snippet: Overexpression of miR-181a promotes the erythroid differentiation of chronic myeloid leukemia K562 cells. The overexpression of miR-181a induced the expression of (A) CD235a and (B) γ-globin in the K562-miR-181a cells (*P

    Article Snippet: Induction and determination of K562 cell differentiation The K562 cells were seeded at a density of 5×105 cells/ml in DMEM, supplemented with 50 µM hemin (Sigma-Aldrich, St. Louis, USA) to induce erythroid differentiation or 25 µM TPA (Sigma-Aldrich) for megakaryocytic differentiation.

    Techniques: Over Expression, Expressing

    Overexpression of miR-181a in chronic myeloid leukemia K562 cells enhanced the chemotherapeutic sensitivity of these cells to the drug imatinib. (A) The overexpression of miR-181a in K562 cells infected with a lentivirus carrying the miR-181a gene was confirmed by reverse transcription-quantitative polymerase chain reaction analysis. K562 cells infected with a lentivirus carrying EGFP were used as negative control. (B) The overexpression of miR-181a in the K562 cells enhanced the inhibitory effect of the chemotherapeutic drug imatinib on the viability of these cells (P

    Journal: Oncology Letters

    Article Title: MicroRNA-181a enhances the chemotherapeutic sensitivity of chronic myeloid leukemia to imatinib

    doi: 10.3892/ol.2015.3663

    Figure Lengend Snippet: Overexpression of miR-181a in chronic myeloid leukemia K562 cells enhanced the chemotherapeutic sensitivity of these cells to the drug imatinib. (A) The overexpression of miR-181a in K562 cells infected with a lentivirus carrying the miR-181a gene was confirmed by reverse transcription-quantitative polymerase chain reaction analysis. K562 cells infected with a lentivirus carrying EGFP were used as negative control. (B) The overexpression of miR-181a in the K562 cells enhanced the inhibitory effect of the chemotherapeutic drug imatinib on the viability of these cells (P

    Article Snippet: Induction and determination of K562 cell differentiation The K562 cells were seeded at a density of 5×105 cells/ml in DMEM, supplemented with 50 µM hemin (Sigma-Aldrich, St. Louis, USA) to induce erythroid differentiation or 25 µM TPA (Sigma-Aldrich) for megakaryocytic differentiation.

    Techniques: Over Expression, Infection, Real-time Polymerase Chain Reaction, Negative Control

    Influence of ATF4 on protease secretion and invasive potential of CML cells and stromal fibroblasts ATF4 was depleted in K562 cells by viral transduction of 4 different clones of shRNA specific to ATF4 (shATF4-c1 – c4); Non-targeting shRNA (shNEG) was used as control A. ATF4 protein level was quantified in whole cell lysates by immunoblot; PARP was used as loading control. B. Protease abundance was measured by antibody array in serum-free conditioned media from cultures of K562 expressing shATF4 (shATF4-c1_S) and control shRNA (shNEG_S). Signal density was analyzed using Image J software and normalized to reference. Graphs are mean protein level ± SEM for n=3 independent samples C. Level of mRNA of selected enzymes was measured by real-time RT-PCR and is shown as mean fold change between K562 cells expressing shATF4-c1 or shATF4-c3 and shNEG ± SEM for n=3 independent samples D. Matrigel invasion of K562 expressing the indicated shRNA constructs was measured using the transwell assay, as in Fig. 2B ; n=3 independent experiments E. Gelatin degradation by HS-5 cells cultured in shATF4-c1_S or shATF4-c3_S and shNEG_S was measured as in Fig. 3E ; n=3. ***p

    Journal: Oncotarget

    Article Title: Increased phosphorylation of eIF2α in chronic myeloid leukemia cells stimulates secretion of matrix modifying enzymes

    doi: 10.18632/oncotarget.12941

    Figure Lengend Snippet: Influence of ATF4 on protease secretion and invasive potential of CML cells and stromal fibroblasts ATF4 was depleted in K562 cells by viral transduction of 4 different clones of shRNA specific to ATF4 (shATF4-c1 – c4); Non-targeting shRNA (shNEG) was used as control A. ATF4 protein level was quantified in whole cell lysates by immunoblot; PARP was used as loading control. B. Protease abundance was measured by antibody array in serum-free conditioned media from cultures of K562 expressing shATF4 (shATF4-c1_S) and control shRNA (shNEG_S). Signal density was analyzed using Image J software and normalized to reference. Graphs are mean protein level ± SEM for n=3 independent samples C. Level of mRNA of selected enzymes was measured by real-time RT-PCR and is shown as mean fold change between K562 cells expressing shATF4-c1 or shATF4-c3 and shNEG ± SEM for n=3 independent samples D. Matrigel invasion of K562 expressing the indicated shRNA constructs was measured using the transwell assay, as in Fig. 2B ; n=3 independent experiments E. Gelatin degradation by HS-5 cells cultured in shATF4-c1_S or shATF4-c3_S and shNEG_S was measured as in Fig. 3E ; n=3. ***p

    Article Snippet: Creation of stable ATF4 knock-down K562 cell line ATF4 was silenced in human K562 cells with MISSION TRC shRNA Lentiviral Particles (#SHCLNVNM_001675/TRCN0000013573, SIGMA); 5 different clones.

    Techniques: Transduction, Clone Assay, shRNA, Ab Array, Expressing, Software, Quantitative RT-PCR, Construct, Transwell Assay, Cell Culture

    Influence of eIF2α phosphorylation on the profile of enzymes secreted by K562 cells A. eIF2α-P level in FACS-sorted K562 expressing GFP alone (K562wt) or GFP and the eIF2α-S51A mutant (K562mut) was analyzed by immunoblot; β-actin is shown as protein loading control. B-C. 18h serum-free conditioned medium cleared by multistep centrifugation from K562wt (K562_S) and K562mut (K562mut_S) was analyzed by quantitative SILAC LC-MS/MS. Pooled results from 3 independent experiments are presented. (B) Volcano plot showing normalized ratio of protein abundance as log2 normalized ratio heavy to light (K562mut_S and K562wt_S) (x-axis) and summed peptides intensities as log2 total intensities (y-axis). (C) Gene Ontology assignment of proteins with significantly different abundance between K562wt_S and K562mut_S to biological processes. D. Abundance of secreted cathepsins (CTS) and matrix metalloproteases (MMP) were analyzed in S-medium by antibody array. Signal density was analyzed using Image J software and normalized to reference. Graphs are mean protein level ± SEM for n=3 independent samples. E. STRING database mapping of proteases identified using SILAC and antibody array. Nodes of clusters are displayed using MCL clustering algorithm with parameter value 2. F. mRNA levels of selected enzymes were quantified by real-time RT-PCR and shown as mean fold change ± SEM for n=3 independent samples of K562mut to K562wt cells ***p

    Journal: Oncotarget

    Article Title: Increased phosphorylation of eIF2α in chronic myeloid leukemia cells stimulates secretion of matrix modifying enzymes

    doi: 10.18632/oncotarget.12941

    Figure Lengend Snippet: Influence of eIF2α phosphorylation on the profile of enzymes secreted by K562 cells A. eIF2α-P level in FACS-sorted K562 expressing GFP alone (K562wt) or GFP and the eIF2α-S51A mutant (K562mut) was analyzed by immunoblot; β-actin is shown as protein loading control. B-C. 18h serum-free conditioned medium cleared by multistep centrifugation from K562wt (K562_S) and K562mut (K562mut_S) was analyzed by quantitative SILAC LC-MS/MS. Pooled results from 3 independent experiments are presented. (B) Volcano plot showing normalized ratio of protein abundance as log2 normalized ratio heavy to light (K562mut_S and K562wt_S) (x-axis) and summed peptides intensities as log2 total intensities (y-axis). (C) Gene Ontology assignment of proteins with significantly different abundance between K562wt_S and K562mut_S to biological processes. D. Abundance of secreted cathepsins (CTS) and matrix metalloproteases (MMP) were analyzed in S-medium by antibody array. Signal density was analyzed using Image J software and normalized to reference. Graphs are mean protein level ± SEM for n=3 independent samples. E. STRING database mapping of proteases identified using SILAC and antibody array. Nodes of clusters are displayed using MCL clustering algorithm with parameter value 2. F. mRNA levels of selected enzymes were quantified by real-time RT-PCR and shown as mean fold change ± SEM for n=3 independent samples of K562mut to K562wt cells ***p

    Article Snippet: Creation of stable ATF4 knock-down K562 cell line ATF4 was silenced in human K562 cells with MISSION TRC shRNA Lentiviral Particles (#SHCLNVNM_001675/TRCN0000013573, SIGMA); 5 different clones.

    Techniques: FACS, Expressing, Mutagenesis, Centrifugation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Ab Array, Software, Quantitative RT-PCR

    Dose- and time-dependent inhibition of K562 cell growth by the lycopene in the free (a) and encapsulated (b) forms. The cells were incubated with increasing concentration of lycopene in the free and encapsulated forms and then the cell survival was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Data were expressed as mean ± standard deviation from three independent experiments (* P

    Journal: Pharmacognosy Magazine

    Article Title: In vitro anti-telomerase activity of novel lycopene-loaded nanospheres in the human leukemia cell line K562

    doi: 10.4103/0973-1296.127368

    Figure Lengend Snippet: Dose- and time-dependent inhibition of K562 cell growth by the lycopene in the free (a) and encapsulated (b) forms. The cells were incubated with increasing concentration of lycopene in the free and encapsulated forms and then the cell survival was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Data were expressed as mean ± standard deviation from three independent experiments (* P

    Article Snippet: Cell culture Human chronic myelogenic leukemia cell line K562 was purchased from Pasteur Institute of Iran (Tehran, Iran).

    Techniques: Inhibition, Incubation, Concentration Assay, Standard Deviation

    The K562 cells were incubated for 48 h without lycopene or with lycopene in the free and encapsulated forms (100 μg/ml) (a) and nuclear morphological changes observed under the fluorescent microscope after staining with hoechst 33342. As compared to control cells (b) fragmented or condensed nuclei indicative of apoptosis could be observed in the lycopene-treated as arrows indicated. The rate of apoptotic K562 cells after treatment with the lycopene in the free (c) and encapsulated (d) forms are presented in the. Data represent as mean ± standard deviation from three independent experiments (* P

    Journal: Pharmacognosy Magazine

    Article Title: In vitro anti-telomerase activity of novel lycopene-loaded nanospheres in the human leukemia cell line K562

    doi: 10.4103/0973-1296.127368

    Figure Lengend Snippet: The K562 cells were incubated for 48 h without lycopene or with lycopene in the free and encapsulated forms (100 μg/ml) (a) and nuclear morphological changes observed under the fluorescent microscope after staining with hoechst 33342. As compared to control cells (b) fragmented or condensed nuclei indicative of apoptosis could be observed in the lycopene-treated as arrows indicated. The rate of apoptotic K562 cells after treatment with the lycopene in the free (c) and encapsulated (d) forms are presented in the. Data represent as mean ± standard deviation from three independent experiments (* P

    Article Snippet: Cell culture Human chronic myelogenic leukemia cell line K562 was purchased from Pasteur Institute of Iran (Tehran, Iran).

    Techniques: Incubation, Microscopy, Staining, Standard Deviation

    Lycopene, in particular in encapsulated forms caused strong apoptotic death on the K562 cell line. Representative Fluorescence Activated Cell Sorting analysis scatter-grams of Annexin V/propidium iodide stained 10, 25, 50, 75, and 100 μg/ml lycopene in the free (a) and loaded (b) forms treatment showed four different cell populations (* P

    Journal: Pharmacognosy Magazine

    Article Title: In vitro anti-telomerase activity of novel lycopene-loaded nanospheres in the human leukemia cell line K562

    doi: 10.4103/0973-1296.127368

    Figure Lengend Snippet: Lycopene, in particular in encapsulated forms caused strong apoptotic death on the K562 cell line. Representative Fluorescence Activated Cell Sorting analysis scatter-grams of Annexin V/propidium iodide stained 10, 25, 50, 75, and 100 μg/ml lycopene in the free (a) and loaded (b) forms treatment showed four different cell populations (* P

    Article Snippet: Cell culture Human chronic myelogenic leukemia cell line K562 was purchased from Pasteur Institute of Iran (Tehran, Iran).

    Techniques: Fluorescence, FACS, Staining