American Society of Hematology

Figure 1

$Figure 1. Effect of BCR-ABL1 kinase inhibition and kinase-inactive BCR-ABL1 on subcellular localization of p27 in CML cells. (A) CML cell lines (K562, KYO-1, and KCL-22) and CD34+ cells from CML patients (N = 11) in CP (N = 8) or BP (N = 3) were treated with imatinib for 16 hours. Cell lines were serum starved in 1% serum and primary samples were cultured in cytokine-free 20% BIT media before performing the experiments. Cytoplasmic (C) and nuclear (N) proteins were fractionated and subjected to immunoblot analysis, using Sp1 and α-tubulin distribution to monitor the purity of the nuclear and cytoplasmic fractions, respectively. Representative immunoblots are shown. Densitometry was performed by normalizing nuclear and cytoplasmic p27 protein levels to levels of Sp1 and α-tubulin, respectively. Expression of untreated cytoplasmic p27 levels was set to 100%. Values represent mean ± SD from 3 independent experiments. *P < .050. (B) Subcellular localization of p27 was analyzed by immunofluorescence (IF) microscopy in CD34+ cells from BM of normal donors or CML patients. Left panel, cells were freshly isolated; right panel, cells were treated with imatinib in cytokine-free 20% BIT media. Representative experiments are shown. Scale bars represent 10 μm. (C) p27−/− MEFs were stably cotransduced with RFP-p27 (wild type) and GFP retroviral vectors containing native BCR-ABL1, the kinase-inactive BCR-ABL1K271R mutant, or empty vector controls. Cells engineered to express native BCR-ABL1 were also treated with 2.5 μM imatinib for 16 hours. Double-positive (GFP+/RFP+) cells were sorted by FACS and the subcellular localization of p27 was visualized by IF using an Alexa Fluor-647–conjugated secondary antibody. Coexpression of GFP and RFP was verified under the microscope. DAPI was used for nuclear staining. Scale bars represent 20 μm. Quantification of nuclear/cytoplasmic p27 signal intensity is shown in supplemental Figure 3. BIT, bovine serum albumin, insulin, and transferrin; DAPI, 4,6 diamidino-2-phenylindole; GFP, green fluorescent protein; RFP, red fluorescent protein.$

Effect of BCR-ABL1 kinase inhibition and kinase-inactive BCR-ABL1 on subcellular localization of p27 in CML cells. (A) CML cell lines (K562, KYO-1, and KCL-22) and CD34⁺ cells from CML patients (N = 11) in CP (N = 8) or BP (N = 3) were treated with imatinib for 16 hours. Cell lines were serum starved in 1% serum and primary samples were cultured in cytokine-free 20% BIT media before performing the experiments. Cytoplasmic (C) and nuclear (N) proteins were fractionated and subjected to immunoblot analysis, using Sp1 and α-tubulin distribution to monitor the purity of the nuclear and cytoplasmic fractions, respectively. Representative immunoblots are shown. Densitometry was performed by normalizing nuclear and cytoplasmic p27 protein levels to levels of Sp1 and α-tubulin, respectively. Expression of untreated cytoplasmic p27 levels was set to 100%. Values represent mean ± SD from 3 independent experiments. *P < .050. (B) Subcellular localization of p27 was analyzed by immunofluorescence (IF) microscopy in CD34⁺ cells from BM of normal donors or CML patients. Left panel, cells were freshly isolated; right panel, cells were treated with imatinib in cytokine-free 20% BIT media. Representative experiments are shown. Scale bars represent 10 μm. (C) p27^−/− MEFs were stably cotransduced with RFP-p27 (wild type) and GFP retroviral vectors containing native BCR-ABL1, the kinase-inactive BCR-ABL1^K271R mutant, or empty vector controls. Cells engineered to express native BCR-ABL1 were also treated with 2.5 μM imatinib for 16 hours. Double-positive (GFP⁺/RFP⁺) cells were sorted by FACS and the subcellular localization of p27 was visualized by IF using an Alexa Fluor-647–conjugated secondary antibody. Coexpression of GFP and RFP was verified under the microscope. DAPI was used for nuclear staining. Scale bars represent 20 μm. Quantification of nuclear/cytoplasmic p27 signal intensity is shown in supplemental Figure 3. BIT, bovine serum albumin, insulin, and transferrin; DAPI, 4,6 diamidino-2-phenylindole; GFP, green fluorescent protein; RFP, red fluorescent protein.

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