Figure 1
Figure 1. Fyn is up-regulated in chronic-phase CML and in BCR-ABL1–expressing cells. (A) Fyn protein expression is increased in blast-crisis CML. A tissue microarray containing bone marrow specimens from 10 chronic-phase patients, 8 accelerated-phase patients, and 16 blastic-phase patients was stained using an anti-Fyn–directed antibody as described in detail in Document S1. The percentage of Fyn-positive cells in each sample was counted and is depicted graphically. Eleven of 16 bone marrow specimens from blast crisis CML patients were 100% positive for Fyn. Numeric percentages next to the horizontal bars refer to average percentage of Fyn-positive cells in each group. (B) Fyn expression increases from chronic phase to blast crisis in the same patient. Paired bone marrow specimens from a patient who progressed from chronic phase to blastic myeloid blast crisis were acquired and stained for Fyn as described in Document S1. Images were acquired on a Nikon Microphot FXA microscope and 20×/0.75 numeric aperture objective lens (total magnification 200×; Nikon Instruments, Melville, NY), with an Olympus DP70 camera (Olympus America, Melville, NY) running DP controller software v.1.2.1.108 (Olympus). Images were processed with Adobe Photoshop CS v.8.0.1 (Adobe Systems, San Jose, CA). (C) Fyn protein levels are increased in BCR-ABL1–expressing cells. Fyn protein was assessed by Western blotting in K562 cells that are imatinib sensitive (lane 1) or imatinib resistant (lane 2), in BaF3 cells transduced with vector (lane 3), wild-type BCR-ABL1 (lane 4), or imatinib-resistant mutant BCR-ABL1 (lanes 5,6) and in parental 32D cells or 32D cells stably transfected with p210 BCR-ABL1. The same membrane was reprobed with antiactin antibody, and the ratio of Fyn/actin protein expression was calculated by densitometry. Numeric values listed below the bands represent a ratio of Fyn protein expression to actin protein expression and are normalized relative to Fyn levels in the BaF3 vector cells, which contain the lowest level of Fyn. Results shown were reproduced in 3 independent experiments. (D) Fyn protein is up-regulated in TonB cells transfected with a tetracycline-inducible BCR-ABL1 expression vector. Western blotting for p210 BCR-ABL1 (top panel) and Fyn (middle panel) in TonB cells transfected with an inducible expression vector after gene induction with 1 μg/mL doxycycline for 12 hours, 24 hours, 36 hours, or 48 hours. Actin protein levels (bottom panel) indicate equal loading. (E) Fyn kinase activity is increased in BCR-ABL1–expressing cells. (Top panel) Immunoprecipitation of Fyn and subsequent Western blotting with a phospho-Src antibody (labeled p-Fyn) or with Fyn antibody (labeled Fyn) shows increased expression of both in BCR-ABL1–expressing cells. Actin was probed in total cell lysates prior to immunoprecipitation. (Bottom panel) Kinase assay for Fyn was conducted in BaF3 vector and BaF3 p210 cells as described in Document S1 using Sam 68 as the substrate. A band corresponding to autophosphorylation of Fyn is also highlighted. Numeric values listed below the bands represent the densitometric analysis of the band corresponding to phosphorylated Sam 68 normalized relative to levels in the BaF3 vector cells (assigned a value of 1). (F) Fyn mRNA is up-regulated in an animal model for CML. Fyn mRNA levels were examined using Fyn-specific primers in cells sorted from a recipient mouse and transplanted with bone marrow from a donor mouse's stem cells transduced with MigR1-GFP-BCR-ABL1. Fyn mRNA was qualitatively measured by RT-PCR in GFP-sorted cells (indicating successful transduction) in the left panel. Also in the left panel, as a positive control for Fyn mRNA expression, BaF3 vector and BaF3 p210 cells were used to show that mRNA for Fyn was increased in BCR-ABL1–expressing cell lines. In the right panel, Fyn mRNA was compared in mice that received a transplant of mouse bone marrow infected with empty MigR1 vector or MigR1 p210-expressing vector. Actin was also amplified. (G) Inhibition of BCR-ABL1 decreases Fyn protein expression. K562 cells were treated with diluent or 0.25 μM imatinib for 24 hours, and levels of Fyn, p210 BCR-ABL1, p145 c-abl, and actin protein were measured by Western blotting. Results are representative of 3 independent experiments.

Fyn is up-regulated in chronic-phase CML and in BCR-ABL1–expressing cells. (A) Fyn protein expression is increased in blast-crisis CML. A tissue microarray containing bone marrow specimens from 10 chronic-phase patients, 8 accelerated-phase patients, and 16 blastic-phase patients was stained using an anti-Fyn–directed antibody as described in detail in Document S1. The percentage of Fyn-positive cells in each sample was counted and is depicted graphically. Eleven of 16 bone marrow specimens from blast crisis CML patients were 100% positive for Fyn. Numeric percentages next to the horizontal bars refer to average percentage of Fyn-positive cells in each group. (B) Fyn expression increases from chronic phase to blast crisis in the same patient. Paired bone marrow specimens from a patient who progressed from chronic phase to blastic myeloid blast crisis were acquired and stained for Fyn as described in Document S1. Images were acquired on a Nikon Microphot FXA microscope and 20×/0.75 numeric aperture objective lens (total magnification 200×; Nikon Instruments, Melville, NY), with an Olympus DP70 camera (Olympus America, Melville, NY) running DP controller software v.1.2.1.108 (Olympus). Images were processed with Adobe Photoshop CS v.8.0.1 (Adobe Systems, San Jose, CA). (C) Fyn protein levels are increased in BCR-ABL1–expressing cells. Fyn protein was assessed by Western blotting in K562 cells that are imatinib sensitive (lane 1) or imatinib resistant (lane 2), in BaF3 cells transduced with vector (lane 3), wild-type BCR-ABL1 (lane 4), or imatinib-resistant mutant BCR-ABL1 (lanes 5,6) and in parental 32D cells or 32D cells stably transfected with p210 BCR-ABL1. The same membrane was reprobed with antiactin antibody, and the ratio of Fyn/actin protein expression was calculated by densitometry. Numeric values listed below the bands represent a ratio of Fyn protein expression to actin protein expression and are normalized relative to Fyn levels in the BaF3 vector cells, which contain the lowest level of Fyn. Results shown were reproduced in 3 independent experiments. (D) Fyn protein is up-regulated in TonB cells transfected with a tetracycline-inducible BCR-ABL1 expression vector. Western blotting for p210 BCR-ABL1 (top panel) and Fyn (middle panel) in TonB cells transfected with an inducible expression vector after gene induction with 1 μg/mL doxycycline for 12 hours, 24 hours, 36 hours, or 48 hours. Actin protein levels (bottom panel) indicate equal loading. (E) Fyn kinase activity is increased in BCR-ABL1–expressing cells. (Top panel) Immunoprecipitation of Fyn and subsequent Western blotting with a phospho-Src antibody (labeled p-Fyn) or with Fyn antibody (labeled Fyn) shows increased expression of both in BCR-ABL1–expressing cells. Actin was probed in total cell lysates prior to immunoprecipitation. (Bottom panel) Kinase assay for Fyn was conducted in BaF3 vector and BaF3 p210 cells as described in Document S1 using Sam 68 as the substrate. A band corresponding to autophosphorylation of Fyn is also highlighted. Numeric values listed below the bands represent the densitometric analysis of the band corresponding to phosphorylated Sam 68 normalized relative to levels in the BaF3 vector cells (assigned a value of 1). (F) Fyn mRNA is up-regulated in an animal model for CML. Fyn mRNA levels were examined using Fyn-specific primers in cells sorted from a recipient mouse and transplanted with bone marrow from a donor mouse's stem cells transduced with MigR1-GFP-BCR-ABL1. Fyn mRNA was qualitatively measured by RT-PCR in GFP-sorted cells (indicating successful transduction) in the left panel. Also in the left panel, as a positive control for Fyn mRNA expression, BaF3 vector and BaF3 p210 cells were used to show that mRNA for Fyn was increased in BCR-ABL1–expressing cell lines. In the right panel, Fyn mRNA was compared in mice that received a transplant of mouse bone marrow infected with empty MigR1 vector or MigR1 p210-expressing vector. Actin was also amplified. (G) Inhibition of BCR-ABL1 decreases Fyn protein expression. K562 cells were treated with diluent or 0.25 μM imatinib for 24 hours, and levels of Fyn, p210 BCR-ABL1, p145 c-abl, and actin protein were measured by Western blotting. Results are representative of 3 independent experiments.

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