Journal: Leukemia

Article Title: Spred1 deficit promotes treatment resistance and transformation of chronic phase CML

doi: 10.1038/s41375-021-01423-x

Figure Lengend Snippet: A Expression of SPRED1 in BM CD34+ cells from patients with BC CML and CP CML by Q-RT-PCR (n=8 samples for BC CML and n=12 samples for CP CML) and western blot and in BM by immunohistochemistry staining (one of three independent experiments with similar results was shown) (left), and expression of miR-126 in CD34+ and CD34+CD38− cells from BC CML (n=6 samples) and CP CML (n=10 samples) patients by Q-RT-PCR (right). B SPRED1 mRNA expression by Q-RT-PCR and protein expression by western blot, miR-126 levels by Q-RT-PCR, cell cycling by Ki-67 and DAPi staining (top) or by cell trace violet staining (bottom) followed by flow cytometry analysis in CML CD34+ cells transduced with SPRED1 siRNA to knock-down (KD) SPRED1 or with a non-targeting control siRNA (Ctrl). UND: undivided cells, G0; DIV: division. C Representative colonies and quantification of colony forming cells (CFC) in CML CD34+ (left) and CD34+CD38− (right) cells transduced with Spred1 siRNA to KD SPRED1 or with ctrl siRNA (n=3). Results shown represent mean ± SEM. Significance values: *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Human CD34 + cells were selected using the indirect CD34 microbead kit (Miltenyi Biotec, San Diego, CA) and CD34 + CD38 − cells were sorted after staining with human antibodies against CD34 and CD38 ( Supplementary Table 1 ) or selected using CD34+CD38- cell isolation kit (Miltenyi Biotec, San Diego, CA) according to the manufacturer’s protocol.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining, Flow Cytometry, Transduction, Knockdown, Control

Journal: Cancers

Article Title: SKF-96365 Expels Tyrosine Kinase Inhibitor-Treated CML Stem and Progenitor Cells from the HS27A Stromal Cell Niche in a RhoA-Dependent Mechanism.

doi: 10.3390/cancers16162791

Figure Lengend Snippet: Figure 1. Imatinib treatment modifies CML-patient CD34+ and Ba/F3p210 cell mobility. (a) CML patients’ CD34+ cells and HS27A cells were stained with 5 µM CFSE and 5 µM CTV, respectively. After 5 h, CFSE-stained CD34+ cells were added to adherent CTV-HS27A cells with or without 5 µM imatinib. After 3 days of co-culture, CFSE-CD34+ and CTV-HS27A were sorted according to CFSE intensity: low CFSE (high proliferation) and high CFSE (low proliferation). (b) The percentage of CD34+ cells hiding under HS27A and the average time spent under HS27A were determined through a time-lapse videomicroscopy analysis (N = 2 patients, n = 5). (c) Time-lapse images (t = 0; t = 3 min; t = 9 min and t = 12 min) extracted from JuliStage recordings of CFSE-stained CD34+ cells from CML patients seeded on CTV-stained HS27A stromal cells in imatinib (5 µM) conditions. Scale bar: 10 µm. Arrowhead shows CD34+ cell hiding under HS27A cells. (d) Cells (0.5 × 105 Ba/F3p210 and Ba/F3p210S509A) were included in 2.5 mg/mL liquid Matrigel diluted in culture medium and cells were incubated with 5 µM imatinib. Each type of displacement (immobile cells, rolling/crawling, or amœboid displacement) was determined through time-lapse videomicroscopy analysis (n > 3). Right panel: spider graph showing the accumulated tracks of analyzed individual cell motility for Ba/F3p210 cells treated or not with imatinib 5 µM. t-test *** p ≤0.001.

Article Snippet: The CD34+ progenitors were sorted with the CD34 Microbead Kit (CD34 Microbead Kit, 130-046-703, Miltenyi Biotec, Paris, France), and the 1–2.105 cells were maintained in StemMACS medium (130-100-473, Miltenyi Biotec, Paris, France) containing rh IL-3 (10 ng/mL—Miltenyi Biotec, Paris, France), rh IL-6 (10 ng/mL—Miltenyi Biotec, Paris, France), rh SCF (25 ng/mL—Miltenyi Biotec, Paris, France), rh TPO (10 ng/mL—Miltenyi Biotec, Paris, France), rh Flt-3 (10 ng/mL—Miltenyi Biotec, Paris, France), and ITS (1/100—Invitrogen, Thermo-Fisher, Illkirch, France) for 4 days in a humidified incubator at 37 ◦C with 5% CO2.

Techniques: Staining, Co-Culture Assay, Incubation

Journal: Cancers

Article Title: SKF-96365 Expels Tyrosine Kinase Inhibitor-Treated CML Stem and Progenitor Cells from the HS27A Stromal Cell Niche in a RhoA-Dependent Mechanism.

doi: 10.3390/cancers16162791

Figure Lengend Snippet: Figure 2. RhoGTPase signaling in imatinib conditions. (a) Western blot analysis of phosphorylated and total Vav, activated (GTP-Rac1), and total Rac1 in Ba/F3p210 cells. Coimmunoprecipitation of Vav and P-Vav, with p210BCR-ABL. Ba/F3p210 cell lysates were immunoprecipitated using anti-ABL IgG and immunoblotted as indicated. Western blot analysis of activated (GTP-Rac1) and total Rac1 in Ba/F3p210 cells obtained via GST-pulldown. (b) Western blot analysis of activated (GTP-RhoA) and total RhoA in Ba/F3p210 cells obtained via GST-pulldown. Affinity-binding assay of phosphorylated and total p210BCR-ABL with negative dominant RhoA (RhoAG17A). Ba/F3p210 cell lysates were immunoprecipitated with sepharose-bound GST-fused with RhoA-G17A and immunoblotted as indicated. (c) Coimmunoprecipitation of ROCK1 and RhoA with p210BCR-ABL. Ba/F3p210 cell lysates were immunoprecipitated with anti-ABL IgG and immunoblotted as indicated. Western blot analysis of phosphorylated MYPT1 and tubulin in Ba/F3p210 cells. (d) Western blot analysis of activated (GTP-RhoA or GTP-Rac1) and total RhoA or Rac1 in CML patient CD34+ cells obtained via GST- pulldown. Western blot analysis of phosphorylated MYPT1 and β-tubulin in CML patient CD34+ cells. Each experiment was carried out 3 times. Ba/F3p210 and CD34+ patient cells were treated with 1 µM ((d) top), 5 µM imatinib (a–c), or as indicated (d).

Article Snippet: The CD34+ progenitors were sorted with the CD34 Microbead Kit (CD34 Microbead Kit, 130-046-703, Miltenyi Biotec, Paris, France), and the 1–2.105 cells were maintained in StemMACS medium (130-100-473, Miltenyi Biotec, Paris, France) containing rh IL-3 (10 ng/mL—Miltenyi Biotec, Paris, France), rh IL-6 (10 ng/mL—Miltenyi Biotec, Paris, France), rh SCF (25 ng/mL—Miltenyi Biotec, Paris, France), rh TPO (10 ng/mL—Miltenyi Biotec, Paris, France), rh Flt-3 (10 ng/mL—Miltenyi Biotec, Paris, France), and ITS (1/100—Invitrogen, Thermo-Fisher, Illkirch, France) for 4 days in a humidified incubator at 37 ◦C with 5% CO2.

Techniques: Western Blot, Immunoprecipitation, Affinity Binding Assay

Journal: Cancers

Article Title: SKF-96365 Expels Tyrosine Kinase Inhibitor-Treated CML Stem and Progenitor Cells from the HS27A Stromal Cell Niche in a RhoA-Dependent Mechanism.

doi: 10.3390/cancers16162791

Figure Lengend Snippet: Figure 3. SKF-96365 induces amœboid movements in immobile imatinib-treated Ba/F3p210 cells. (a) Cells (0.5 × 105 Ba/F3p210 and Ba/F3p210S509A) were included in 2.5 mg/mL liquid Matrigel diluted in culture medium and incubated with or without 5 µM imatinib and/or 40 µM SKF-96365. Each type of displacement (immobile cells, rolling/crawling, or amœboid displacement) was determined through time-lapse videomicroscopy analysis. The right panel shows the analysis of amoeboid contractions in immobile cells (n > 3). t-test ** p ≤0.01; *** p ≤0.001; ns: non-significant. (b) Western blot analysis of phosphorylated and total p210BCR-ABL in Ba/F3p210 cells during imatinib and SKF-96365 treatment. (c) Western blot analysis of phosphorylated and total Vav, activated (GTP- Rac1), and total Rac1 in Ba/F3p210 cells during imatinib and SKF-96365 treatment. (d) Upper panel: Western blot analysis of activated (GTP-RhoA) and total RhoA or Rac1 in Ba/F3p210 cells obtained via GST-pulldown. Lower panel: Western blot analysis of phosphorylated MYPT1 and β-tubulin in CML patient CD34+ cells. (e) Coimmunoprecipitation of RhoA with p210BCR-ABL. Ba/F3p210 cell lysates were immunoprecipitated with anti-ABL IgG and immunoblotted as indicated.

Article Snippet: The CD34+ progenitors were sorted with the CD34 Microbead Kit (CD34 Microbead Kit, 130-046-703, Miltenyi Biotec, Paris, France), and the 1–2.105 cells were maintained in StemMACS medium (130-100-473, Miltenyi Biotec, Paris, France) containing rh IL-3 (10 ng/mL—Miltenyi Biotec, Paris, France), rh IL-6 (10 ng/mL—Miltenyi Biotec, Paris, France), rh SCF (25 ng/mL—Miltenyi Biotec, Paris, France), rh TPO (10 ng/mL—Miltenyi Biotec, Paris, France), rh Flt-3 (10 ng/mL—Miltenyi Biotec, Paris, France), and ITS (1/100—Invitrogen, Thermo-Fisher, Illkirch, France) for 4 days in a humidified incubator at 37 ◦C with 5% CO2.

Techniques: Incubation, Western Blot, Immunoprecipitation

Journal: Cancers

Article Title: SKF-96365 Expels Tyrosine Kinase Inhibitor-Treated CML Stem and Progenitor Cells from the HS27A Stromal Cell Niche in a RhoA-Dependent Mechanism.

doi: 10.3390/cancers16162791

Figure Lengend Snippet: Figure 5. SKF-96365 expels low-proliferating CD34+ cells from the HS27A niche. (a) CML patient CD34+ cells and HS27A cells were stained with 5 µM CFSE and 5 µM CTV, respectively. After 5 h, CFSE-stained CD34+ cells were added to adherent CTV-HS27A cells with or without 5 µM imatinib. After 3 days of co-culture, CFSE-CD34+ and CTV-HS27A were sorted according to CFSE intensity: low CFSE (high proliferation) and high CFSE (low proliferation). CFSE-sorted CD34+ cells from CML patients were reseeded on previously cultured CTV-stained HS27A cells and cultured for 2 days under the indicated conditions. Then, videomicroscopy was performed using JuliStage in fluorescent mode during 6 h recording. (b) The percentage of CD34+ cells hiding under HS27A cells and the average time spent under HS27A cells were determined through time-lapse videomicroscopy analysis (N = 2 patients, n = 5). (c) Analysis of the hiding behavior of low-proliferating CD34+ cells from CML patients during the recording time. Two behaviors were identified corresponding to a short-duration stay under HS27A cells (SD; <30 min) and a long-duration stay (LD; 30 to >300 min) (see Supplementary Figure S5 for details).

Article Snippet: The CD34+ progenitors were sorted with the CD34 Microbead Kit (CD34 Microbead Kit, 130-046-703, Miltenyi Biotec, Paris, France), and the 1–2.105 cells were maintained in StemMACS medium (130-100-473, Miltenyi Biotec, Paris, France) containing rh IL-3 (10 ng/mL—Miltenyi Biotec, Paris, France), rh IL-6 (10 ng/mL—Miltenyi Biotec, Paris, France), rh SCF (25 ng/mL—Miltenyi Biotec, Paris, France), rh TPO (10 ng/mL—Miltenyi Biotec, Paris, France), rh Flt-3 (10 ng/mL—Miltenyi Biotec, Paris, France), and ITS (1/100—Invitrogen, Thermo-Fisher, Illkirch, France) for 4 days in a humidified incubator at 37 ◦C with 5% CO2.

Techniques: Staining, Co-Culture Assay, Cell Culture

Journal:

Article Title: Human C/EBP-? activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation

doi: 10.1182/blood-2008-02-139741

Figure Lengend Snippet: Temporal changes in expression of the C/EBP-ϵ isoforms during eosinophilopoiesis. CD34+ progenitors were differentiated to the eosinophil lineage by suspension culture in SCF, IL-3, IL-5, GM-CSF, and Flt3-L for 3 days, followed by only IL-3 and IL-5 thereafter. The cells were maintained at 0.5 × 106 cells/mL, total and eosinophil counts determined every 3 to 4 days, and total RNA for RT-PCR and total protein for Western blotting prepared from 1 × 106 cells. Cell proliferation (A) and the percentage of eosinophils (B) developing in the cultures based on differential cell counts using Fast Green/Neutral Red staining to distinguish secondary granule formation is shown. Semiquantitative RT-PCR was performed using α-32P-dCTP and C/EBP-ϵ isoform selective primers, with β2M amplified as the internal control for mRNA (cDNA) input (C). C/EBP-ϵ isoform protein expression was analyzed by Western blotting of whole cell lysates using a combination of anti–C/EBP-ϵ C-terminal (C-22; SC-158) and N-terminal (H-75; SC-25770) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), compared with GAPDH expression as the loading control (D). Representative results from 2 independent experiments are shown.

Article Snippet: After 72 hours, the cells were stained with anti–CD34-phycoerythrin (PE)–conjugated antibody or IgG1 isotype control (BD PharMingen, San Diego, CA) and sorted by FACS (MoFlo; Dako North America, Carpinteria, CA) based on dual expression of CD34 and GFP.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Amplification

Journal:

Article Title: Human C/EBP-? activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation

doi: 10.1182/blood-2008-02-139741

Figure Lengend Snippet: Effects of the C/EBP-ϵ isoforms on myeloid and erythroid lineage colony formation. CD34+ CB progenitors were transduced with retroviral vectors encoding the C/EBP-ϵ isoforms and after 72 hours of transduction, CD34+GFP+ cells were sorted by FACS and cultured in semisolid Collagen Cult (StemCell Technologies) colony assays using cytokines to drive differentiation of mixed myeloid lineages excluding erythroid (A: culture in SCF, GM-CSF, IL-3, and IL-5), or both mixed myeloid and erythroid lineages (B: culture with SCF, GM-CSF, IL-3, IL-5, and EPO). The mean (± SEM) number of granulocyte-macrophage colony-forming unit (CFU-GM), granulocyte-erythrocyte-macrophage-megakaryocyte colony-forming unit (CFU-GEMM), and BFU-E colonies that developed from 750 plated CD34+GFP+ transduced progenitors is plotted for 3 or 4 independent experiments performed in duplicate or triplicate. Statistically significant differences of interest are shown (brackets and P values) for comparisons using 1-way analysis of variance (ANOVA) and least significant difference (LSD).

Article Snippet: After 72 hours, the cells were stained with anti–CD34-phycoerythrin (PE)–conjugated antibody or IgG1 isotype control (BD PharMingen, San Diego, CA) and sorted by FACS (MoFlo; Dako North America, Carpinteria, CA) based on dual expression of CD34 and GFP.

Techniques: Transduction, Cell Culture

Journal:

Article Title: Human C/EBP-? activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation

doi: 10.1182/blood-2008-02-139741

Figure Lengend Snippet: Effects of the C/EBP-ϵ isoforms on eosinophil, erythroid, and neutrophil differentiation. CD34+ CB progenitors were transduced with retroviral vectors encoding each of the C/EBP-ϵ isoforms. After 72 hours of transduction, CD34+GFP+ cells were sorted by FACS, plated in Collagen Cult colony assays, and the cells induced to differentiate toward the eosinophil (A: culture in SCF, IL-3, and IL-5), neutrophil (B: culture in SCF, IL-3, and G-CSF), and erythroid (C: culture in SCF, IL-3, and EPO) lineages. The mean (± SD) number of GM, eosinophil, and erythroid colonies that developed from 750 plated CD34+GFP+ transduced progenitors is plotted for 3 independent experiments performed in duplicate or triplicate. The effect of the C/EBP-ϵ isoforms on plating efficiency is shown in panel D as the total number of hematopoietic colonies (myeloid + erythroid) developed from 500 plated CD34+GFP+ progenitors for each of the cytokine cocktails used to induce neutrophil (G-CSF), eosinophil (IL-5), and erythroid (EPO) colony formation. Statistically significant differences are shown for comparisons using 1-way ANOVA and LSD (*P ≤ .05, ***P ≤ .001).

Article Snippet: After 72 hours, the cells were stained with anti–CD34-phycoerythrin (PE)–conjugated antibody or IgG1 isotype control (BD PharMingen, San Diego, CA) and sorted by FACS (MoFlo; Dako North America, Carpinteria, CA) based on dual expression of CD34 and GFP.

Techniques: Transduction

Journal:

Article Title: Human C/EBP-? activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation

doi: 10.1182/blood-2008-02-139741

Figure Lengend Snippet: C/EBP-ϵ32/30 enhancement of eosinophil differentiation does not require IL-5. CD34+ CB progenitors were transduced 3 times over a period of 72 hours with the retroviral vector encoding the C/EBP-ϵ32 activator isoform or empty vector control. CD34+GFP+ cells were sorted by FACS, and either plated in Collagen Cult colony assay media containing SCF and IL-3, and the cells allowed to differentiate for 14 days without (A) or with (B) IL-5 added to drive eosinophil colony formation. Eosinophil and GM colonies were enumerated using staining with Fast Green/Neutral Red and May-Grünwald Giemsa, respectively. The mean (± SD) number of CFU-Eos and CFU-GM colonies that developed from 750 CD34+GFP+ transduced progenitors is plotted for triplicate determinations (A,B).

Article Snippet: After 72 hours, the cells were stained with anti–CD34-phycoerythrin (PE)–conjugated antibody or IgG1 isotype control (BD PharMingen, San Diego, CA) and sorted by FACS (MoFlo; Dako North America, Carpinteria, CA) based on dual expression of CD34 and GFP.

Techniques: Plasmid Preparation, Colony Assay, Staining

Journal:

Article Title: Human C/EBP-? activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation

doi: 10.1182/blood-2008-02-139741

Figure Lengend Snippet: The C/EBP-ϵ isoforms differentially induce or inhibit eosinophil gene transcription. Analysis by real-time RT-Q-PCR. CD34+ CB cells were transduced 3 times with the retroviral vectors encoding the 3 C/EBP-ϵ isoforms and empty retroviral vector control over a period of 72 hours, double CD34+GFP+ progenitors sorted by FACS, grown for 14 days in suspension cultures supplemented with IL-3 + IL-5 to drive eosinophil differentiation (as in Figure 6), and total RNA prepared and reverse-transcribed to cDNA. The expression of genes encoding eosinophil secondary granule proteins, including major basic protein-1 (MBP1), eosinophil-derived neurotoxin/ribonuclease-2 (EDN, RNS2), and eosinophil peroxidase (EPX) (A), the soluble and transmembrane alternative RNA splice forms of the eosinophil-specific IL-5 receptor α (IL-5Rα) subunit (B), and GATA-1 and β-globin (C) were analyzed by RT-Q-PCR. The mean cDNA expression levels relative to the expression of the β2M input control in each sample amplified at the same time are plotted (± SD) for 3 independent experiments analyzed in triplicate. Statistically significant differences are shown for comparisons between means using 1-way ANOVA and LSD (*P ≤ .05, ***P ≤ .001).

Article Snippet: After 72 hours, the cells were stained with anti–CD34-phycoerythrin (PE)–conjugated antibody or IgG1 isotype control (BD PharMingen, San Diego, CA) and sorted by FACS (MoFlo; Dako North America, Carpinteria, CA) based on dual expression of CD34 and GFP.

Techniques: Plasmid Preparation, Expressing, Derivative Assay, Amplification

Journal:

Article Title: Human C/EBP-? activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation

doi: 10.1182/blood-2008-02-139741

Figure Lengend Snippet: Transduction of CD34+ progenitors with the C/EBP-ϵ32/30 activator isoforms blocks expression of the C/EBP-ϵ14 repressor isoform. CD34+ cells were transduced for 72 hours with the C/EBP-ϵ32/30 (lane 1) or empty GFP (lane 2) retroviral vectors and grown in suspension cultures supplemented with IL-5 to drive eosinophil differentiation. Total protein and RNA was prepared at 14 days from 106 cells lysed in TRIzol (Invitrogen) and analyzed by Western blotting (A) for expression of the C/EBP-ϵ32/30 and ϵ14 isoforms, or semiquantitative RT-PCR (B) for expression of the C/EBP-ϵ14 repressor isoform. Western blotting for GAPDH was used to control for equal protein loading (A), and PCR for β2M was used for comparison of cDNA inputs (B). Vertical lines have been inserted in panel A to indicate repositioned gel lanes.

Article Snippet: After 72 hours, the cells were stained with anti–CD34-phycoerythrin (PE)–conjugated antibody or IgG1 isotype control (BD PharMingen, San Diego, CA) and sorted by FACS (MoFlo; Dako North America, Carpinteria, CA) based on dual expression of CD34 and GFP.

Techniques: Transduction, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction