Journal: Leukemia

Article Title: P-glycoprotein-mediated drug efflux is a resistance mechanism of chronic myelogenous leukemia cells to treatment with imatinib mesylate.

doi: 10.1038/sj.leu.2403257

Figure Lengend Snippet: Figure 1 Description of the MDR phenotype in the investigated cell lines. (a) The expression pattern of potential candidates for an MDR phenotype-related protein in the investigated cell lines CCRF, HL-60, K562 and their respective drug-resistant counterparts (level of resistance is indicated – italic letters). Cells were maintained under routine conditions (sensitive cell lines) or in the presence of the indicated drug (CCRF-VCR – 0.1 mM vincristin, HL-60 and K562–1 mM, and/or 5 mM of VP16, respectively). Western blot conditions were as described in the Materials and methods. Membranes were loaded with equal amounts of protein and were exposed to the film in parallel. Therefore, expression patterns reflect relative amounts of expression (between sensitive and resistant cell lines, and between the different cell lines used). Actin controls are shown in the bottom to ensure equal loading of protein in each lane. One representative determination was chosen out of three with equivalent results. (b) Rhodamin efflux from the sensitive K562 cell line and their respective multidrug-resistant counterparts. Cell lines were analyzed as it is described in the Materials and methods comparing intracellular rhodamin staining in samples treated with rhodamin alone (black) or after the coadminis- tration of 5 mM CsA þ rhodamin (gray). (c) The expression pattern of one critical downstream effector of Bcr-Abl in CCRF-CEM, HL-60 and K562 cells, and their resistant counterparts. P-Crkl denotes the phosphorylated variant of the Crkl protein. Whole Crkl antigen was detected to assure equal loading in the lane.

Article Snippet: Cell lines, kinase inhibitor and drugs The parental HL-60, CCRF-CEM and K562 cell lines were obtained from DSMZ (DSMZ, Braunschweig, Germany).

Techniques: Expressing, Western Blot, Staining, Variant Assay

Journal: Leukemia

Article Title: P-glycoprotein-mediated drug efflux is a resistance mechanism of chronic myelogenous leukemia cells to treatment with imatinib mesylate.

doi: 10.1038/sj.leu.2403257

Figure Lengend Snippet: Figure 3 Intracellular imatinib levels in K562 and drug-resistant K562 cells expressing different amounts of Pgp. (a) The determination of intracellular imatinib levels in parental K562 cells and in drug- resistant K562–1 mM VP16 and K562–5 mM VP16 cell lines. Cells were either treated with 5 mg/ml (8.5 mM) imatinib alone (a) or in combination with 5 mM CsA (b) for 5 h. The means of two independent experiments that were determined in triplet each is indicated by a horizontal bar; s.d. is indicated. Determinations in resistant K562–1 mM VP16 cells (J) were analyzed in parallel with sensitive K562 cells (&) at the corresponding day; resistant K562–5 mM cells (n) were analyzed in another independent determination with the corresponding sensitive K562 cell line (B). The determination of the ratio between intracellular imatinib levels detected in K562–1 mM VP16 cells, K562– 5 mM VP16 cells vs K562-sensitive cells at the corresponding day. Cells were either treated with imatinib 5 mg/ml (8.5 mM) (a) or treated with the combination of imatinib 5 mg/ml (8.5 mM) and CsA 5 mM (b). MTT analysis of K562-sensitive, K562–1 mM VP16 and K562–5 mM VP16 cells that were exposed to different concentrations of imatinib. Cells were exposed for 48 h and MTT was performed as it is described in the Materials and methods. Results resemble the means of three independent experiments that were determined in triplet each. OD values of untreated control cells were set 100%. All other values refer to the untreated control.

Article Snippet: Cell lines, kinase inhibitor and drugs The parental HL-60, CCRF-CEM and K562 cell lines were obtained from DSMZ (DSMZ, Braunschweig, Germany).

Techniques: Expressing, Control

Journal: Leukemia

Article Title: P-glycoprotein-mediated drug efflux is a resistance mechanism of chronic myelogenous leukemia cells to treatment with imatinib mesylate.

doi: 10.1038/sj.leu.2403257

Figure Lengend Snippet: Figure 4 Modulation of Pgp function is restoring apoptotic response after STI571 treatment in resistant K562 cells. The effect of Pgp modulation treatment on phosphorylation pattern of Crkl. Sensitive and resistant K562 cell lines were treated either with CsA (5 mM) alone for 3 h, imatinib (5 mM) alone for 2.5 h or were pre- exposed to CsA (5 mM) 30 min and then coincubated with imatinib (5 mM) for additional 2.5 h. Thereafter, equal amounts of protein (40 mg) were analyzed by immunoblotting against p-Crkl (where indicated). The blots were stripped and reprobed against Crkl (where indicated). The effect of CsA cotreatment on apoptotic cell death as determined by Annexin V staining. Sensitive and resistant K562 cell lines were treated either with CsA (5 mM) alone for 48 h, imatinib (1 mM) alone for 48 h or were pre-exposed to CsA (5 mM) 30 min and then coincubated with imatinib (1 mM) for additional 48 h. Values represent the means of three independent determinations 7s.d.

Article Snippet: Cell lines, kinase inhibitor and drugs The parental HL-60, CCRF-CEM and K562 cell lines were obtained from DSMZ (DSMZ, Braunschweig, Germany).

Techniques: Phospho-proteomics, Western Blot, Staining

Journal: Leukemia

Article Title: P-glycoprotein-mediated drug efflux is a resistance mechanism of chronic myelogenous leukemia cells to treatment with imatinib mesylate.

doi: 10.1038/sj.leu.2403257

Figure Lengend Snippet: Figure 5 Effect of a CsA modulated imatinib therapy in a patient with relapsed BCR-ABL-positive ALL. (a) Rhodamin efflux in patient 01/03 blast samples. Cells were analyzed after a routine Ficoll isolation procedure as it is described in Materials and Methods comparing intracellular rhodamin staining in samples treated with rhodamin alone (black area) or after the coadministration of 5 mM CsA þ rhodamin (grey curve). (b) The treatment schedule of the patient 01/03 as compared to the blast amount in peripheral blood. (c) The phosphorylation pattern of Crkl in a patient treated with a combined treatment approach. Blast cells were analyzed while treating the patient with 800 mg imatinib/day (day 0) and 24 h after starting high- dose CsA administration þ imatinib (day 1). Protein probes of patient 01/03 blasts were obtained as it is described in Materials and methods. Thereafter, equal protein amounts (40 mg) were analyzed by immuno- blotting against p-Crkl (where indicated). The blots were stripped and reprobed against Crkl (where indicated). K562 cells with previously shown strong phosphorylation of Crkl served as control.

Article Snippet: Cell lines, kinase inhibitor and drugs The parental HL-60, CCRF-CEM and K562 cell lines were obtained from DSMZ (DSMZ, Braunschweig, Germany).

Techniques: Isolation, Staining, Phospho-proteomics, Control

Journal: Scientific Reports

Article Title: Down syndrome-associated haematopoiesis abnormalities created by chromosome transfer and genome editing technologies

doi: 10.1038/srep06136

Figure Lengend Snippet: (a) Sequence analyses of GATA1s-ES cells. One allele had an 8-bp deletion and the other had a 17-bp deletion; both resulted in a TGA stop codon in exon 2 of GATA1 . The GATA1 nucleotide (upper line) and amino acid (lower line) sequences are shown for WT-ES and GATA1s-ES cells. An asterisk shows the stop codon. (b) Western blot analyses of erythroid lineage-differentiated cells derived from WT-ES, WT-ES sublines, Ts21-ES lines, GATA1s-ES, and GATA1s/Ts21-ES lines. Anti-BACH1 was used to detect the gene-dosage effect on hChr.21. Anti-GATA1 recognised the C-terminus of both GATA1 and GATA1s protein. Anti-α-tubulin was used as an internal control. HEL cell lysate and K562 nuclear extract were used as positive controls. 10T1/2 whole cell lysate was used as a negative control. Cropped blots were used in this figure. Original full-length blots are shown in . (c, d) Haematopoietic differentiation analyses using WT-ES, WT-ES sublines, Ts21-ES lines, GATA1s-ES, and GATA1s/Ts21-ES lines. Data are the means of 3 independent experiments (±S.D.). The percentage of CD34+, CD41a+/CD42b+, and CD71+/CD235+ cells are shown in each differentiation stage (ES-sac (day 14), megakaryocyte (day 20), and erythroid (day 20)) (c). Statistical analyses were performed by comparison with WT-ES cells (WT-ES1, WT-ES1-1 and WT-ES1-2). *p < 0.05, **p < 0.01 by two-tailed Student's t test. The percentage of CD34-/CD41a-, CD34+/CD41a+ and CD34-/CD41a+ cells are shown in the megakaryocyte stage (d).

Article Snippet: HEL 92.1.7 whole cell lysate and K562 nuclear extract were used as positive controls (sc-2130 and sc-2277, respectively; Santa Cruz Biotechnology).

Techniques: Sequencing, Western Blot, Derivative Assay, Control, Negative Control, Comparison, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Cooperation between Hsp90 and mortalin/GRP75 in resistance to cell death induced by complement C5b-9

doi: 10.1038/s41419-017-0240-z

Figure Lengend Snippet: a , b K562 cells were incubated with geldanamycin (GA, 100 μM) ( a ) or radicicol (Rad, 100 μM) or with DMSO (0.5%) as control for 60 min at 37 °C. Then, the cells were washed and treated with antibody and NHS for 60 min at 37 °C. Percent lysis was determined by propidium iodide inclusion. c Ramos cells were incubated with geldanamycin (GA, 100 μM) or with DMSO (0.5%) as control for 60 min at 37 °C. Then, the cells were washed and treated with rituximab (3 μg/ml) and NHS (50%) for 60 min at 37 °C. Percent lysis was determined by propidium iodide inclusion. d K562 cells were treated with geldanamycin or with DMSO for 60 min, followed by antibody (30 min) and NHS (10 min, peak C5b-9 formation). The cells were then treated or not with trypsin and labeled with aE11 monoclonal antibody followed by a secondary FITC-labeled antibody. Mean fluorescence intensity (MFI) of bound C5b-9, representative of three independent experiments, is presented. * P < 0.05, ** P < 0.01

Article Snippet: K562 cell lysates were first treated overnight with protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) in TBS at 4 °C.

Techniques: Incubation, Control, Lysis, Labeling, Fluorescence

Journal: Cell Death & Disease

Article Title: Cooperation between Hsp90 and mortalin/GRP75 in resistance to cell death induced by complement C5b-9

doi: 10.1038/s41419-017-0240-z

Figure Lengend Snippet: a – d Hsp90β-C9 binding was tested by co-sedimentation through sucrose gradients. Recombinant Hsp90β was incubated with C9 (1 µg each) for 1 h at 37 °C. The samples were layered on top of 13 ml of 10–30% sucrose density gradients that were subjected to unltracentrifugation for 18 h at 40,000 rpm. Fractions (300 µl) were collected from the gradient top. Samples (90 µl) from each fraction were analyzed by dot blotting with anti-Hsp90 ( a , b ) or anti-C9 ( c , d ) antibody and a peroxidase-conjugated secondary antibody. Representative dot blots are shown ( a , c ). Density of each scanned dot was quantified in arbitrary units (A.U.). Relative distribution of Hsp90 ( b ) and C9 ( d ) in fractions 1–24 of the gradients is shown. e , f Microtiter plate wells were coated with recombinant C9 or BSA as control. Then, K562 cells lysates ( e ) or recombinant Hsp90β ( f ) were added to the wells for 60 min at 37 °C. Binding of cytosolic ( e ) and recombinant ( f ) Hsp90 was quantified (optical density, A450) with anti-Hsp90 antibodies, followed by peroxidase-conjugated secondary antibodies. Increasing quantities of C9 yielded higher Hsp90 binding ( P < 0.001, one-way ANOVA)

Article Snippet: K562 cell lysates were first treated overnight with protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) in TBS at 4 °C.

Techniques: Binding Assay, Sedimentation, Recombinant, Incubation, Control

Journal: Cell Death & Disease

Article Title: Cooperation between Hsp90 and mortalin/GRP75 in resistance to cell death induced by complement C5b-9

doi: 10.1038/s41419-017-0240-z

Figure Lengend Snippet: a Hsp90 inhibits C9 polymerization. C9 (1 µg) was mixed with recombinant Hsp90β or heat (60 °C) denatured Hsp90β (dHSP90), or BSA as control (1 µg) and then incubated with or without ZnCl2 for 2 h at 37 °C. The samples were subjected to SDS-PAGE on a 3–10% acrylamide gradient gel and stained with Coomassie blue. The bands of poly C9, monomeric C9, and Hsp90β are indicated. b Cell lysates after complement activation contain Hsp90–C9 complexes. K562 cells were incubated with a sublytic dose of anti-K562 antibody and with normal human serum (NHS) or heat-inactivated (HI) serum for 10 min at 37 °C. Cell lysates were prepared and then immunoprecipitated with mouse anti-Hsp90 antibody (or control mouse IgG) coupled to agarose beads. Proteins attached to the beads were examined by SDS-PAGE and western blotting. Detection was with anti-Hsp90 (upper) and anti-C9 (lower) antibodies

Article Snippet: K562 cell lysates were first treated overnight with protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) in TBS at 4 °C.

Techniques: Recombinant, Control, Incubation, SDS Page, Staining, Activation Assay, Immunoprecipitation, Western Blot

Journal: Cell Death & Disease

Article Title: Cooperation between Hsp90 and mortalin/GRP75 in resistance to cell death induced by complement C5b-9

doi: 10.1038/s41419-017-0240-z

Figure Lengend Snippet: a , b K562 cells were transfected with mortalin siRNA (Mot) or a non-specific siRNA (SC). After 48 h, the level of mortalin in the cells was examined by SDS-PAGE and western blotting with anti-mortalin antibody and with anti-actin antibodies ( a ). Transfected cells were treated with GA (100 μM) or DMSO and then subjected to treatment with antibody and NHS. Percent lysis was determined by propidium iodide inclusion ( b ). NT non-treated. c , d Microtiter plate wells were coated with recombinant mortalin (C) or Hsp90β (D) or BSA as control (0). Then, K562 cells lysates ( c ) or recombinant mortalin ( d ) were added to the wells for 60 min at 37 °C. Binding of Hsp90 ( c ) was quantified (optical density, A450) with anti-Hsp90 antibodies and binding of His-tagged mortalin ( d ) was detected with anti-His antibodies, both followed by peroxidase-conjugated secondary antibodies. A dose-dependent binding of Hsp90 and mortalin was observed ( P < 0.05 ( c ), P < 0.001 ( d ), one-way ANOVA). e K562 cells were incubated with a sublytic dose of anti-K562 antibody and with normal human serum (NHS) or heat-inactivated (HI) serum for 10 min at 37 °C. Cell lysates were prepared and then immunoprecipitated with mouse anti-Hsp90 antibody (or control mouse IgG) coupled to agarose beads. Proteins attached to the beads were examined by SDS-PAGE and western blotting. Detection was with anti-Hsp90 (upper) or anti-mortalin (lower) antibodies. f Microtiter plate wells were coated with C9 (100 ng/well) overnight. Mortalin was mixed with Hsp90β or BSA and added to the wells. Binding of His-tagged mortalin to C9 was quantified with anti-His (mortalin) antibodies and peroxidase-conjugated secondary antibodies. Mortalin–C9 binding was significantly inhibited by Hsp90β but not by BSA ( P < 0.001, two-way ANOVA)

Article Snippet: K562 cell lysates were first treated overnight with protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) in TBS at 4 °C.

Techniques: Transfection, SDS Page, Western Blot, Lysis, Recombinant, Control, Binding Assay, Incubation, Immunoprecipitation

Journal: Biochimica et biophysica acta

Article Title: Imatinib restores VASP activity and its interaction with Zyxin in BCR-ABL leukemic cells.

doi: 10.1016/j.bbamcr.2014.11.008

Figure Lengend Snippet: Fig. 1. VASP and Zyxin modulate apoptosis-related proteins. (A–D) Evaluation of VASP and Zyxin silencing in K562 cells transduced with lentivirus-mediated shRNA control and lentivirus mediated shRNA targeting VASP and Zyxin by quantitative RT-PCR analysis (A, C) and Western blotting analysis (B, D), respectively. (E–G) Western blotting analysis was used for quantification of protein expression and activity. Respective total protein or Actin was used as a control to ensuring equal sample loading; the antibodies used for immunoblotting (IB) are indicated.

Article Snippet: Briefly, 500 μg of total K562 cell extracts was incubated overnight with 20 μL anti-VASP antibody (Santa Cruz Biotechnology) or with normal goat immunoglobulin (IgG) as a negative control.

Techniques: Transduction, shRNA, Control, Quantitative RT-PCR, Western Blot, Expressing, Activity Assay

Journal: Biochimica et biophysica acta

Article Title: Imatinib restores VASP activity and its interaction with Zyxin in BCR-ABL leukemic cells.

doi: 10.1016/j.bbamcr.2014.11.008

Figure Lengend Snippet: Fig. 3. VASP and Zyxin silencing does not modulate proliferation and clonogenicity. (A) and (B) Cell proliferation/viability was determined by MTT assay of incubation of shControl or shVASP or shZyxin K562 cells, respectively, treated or not with imatinib mesylate (0.1; 0.5 or 1 μM) after 48 h and normalized by untreated shControl cells. Values are expressed in per- centage, and normalized to the shControl value set as 100%. Results are shown as mean ± SD of at least three independent experiments. The P values are indicated in the figure; two-way ANOVA test and Bonferroni post-test (C) and (D) shControl or shVASP or shZyxin cells were cultured in methyl cellulose and colonies were detected by MTT after 8 days of incubation with or without treated imatinib mesylate (0.1; 0.5 or 1 μM) and were normalized by untreated shControl cells. Values are expressed in percentage, and normalized to the shControl value set as 100%. Colony images are representative of one experiment and the bar graphs are shown as mean ± SD of at least three independent experiments.

Article Snippet: Briefly, 500 μg of total K562 cell extracts was incubated overnight with 20 μL anti-VASP antibody (Santa Cruz Biotechnology) or with normal goat immunoglobulin (IgG) as a negative control.

Techniques: MTT Assay, Incubation, Cell Culture

Journal: Biochimica et biophysica acta

Article Title: Imatinib restores VASP activity and its interaction with Zyxin in BCR-ABL leukemic cells.

doi: 10.1016/j.bbamcr.2014.11.008

Figure Lengend Snippet: Fig. 4. Imatinib treatment enhances VASP and Zyxin interaction, increases VASP phosphorylation at serine 157 and reduces association of VASP and BCR–ABL. (A) The same lysates from K562 cells untreated and treated with Imatinib (1 μM) were immunoprecipitated (IP) with anti-Zyxin antibody and immunoblotted (IB) with anti-Zyxin and anti-VASP antibodies. Total protein extract (input) and isotype IgG antibody were used as controls.(B) Western-blotting using anti-p-VASP ser157, anti-VASP and Actin antibodies in K562 cells treated with Imatinib (1 μM) for 3, 6, 9 and 12 h. (C) Endogenous VASP were co-immunoprecipitated with BCR–ABL. Total extracts from K562 cells treated with Imatinib (1 μM) were submitted to immuno- precipitation with an anti-VASP antibody followed by western blot analysis using anti-ABL and anti-VASP antibody. Total protein extract (input) and isotype IgG antibody were used as controls. (D) Western blotting showed phospho-VASP ser 157, VASP, phospho-Zyxin ser 142 and Zyxin in K562 cells, one healthy donor, one CML patient at diagnosis, one responding and two resistant to Imatinib. Actin staining indicates the amount of protein loaded. All images in Fig. 4E belong to the same western blotting and the membrane was stripped and reprobed with different antibodies. Actin was used as a control to ensuring equal sample loading; the antibodies used for immunoblotting (IB) are indicated.

Article Snippet: Briefly, 500 μg of total K562 cell extracts was incubated overnight with 20 μL anti-VASP antibody (Santa Cruz Biotechnology) or with normal goat immunoglobulin (IgG) as a negative control.

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Biomarker Discovery, Staining, Membrane, Control