American Society of Hematology

Figure 3

$Figure 3. WP1130 induces cellular trafficking of Bcr-Abl and inhibits its substrate phosphorylation in CML cells. (A) BV-173 cells were treated with 5μM WP1130 for the intervals indicated before assessing Bcr-Abl and other protein levels in total cell lysates and in the detergent-soluble and -insoluble fractions. Actin was blotted as a protein-loading control. (B) BV-173 and BV-173R cells were treated with 5μM WP1130 for 2 hours before detergent-soluble and -insoluble cell lysates were blotted for Bcr-Abl (K12 antibody) and actin as a protein-loading control. (C) K562R cells were treated with 5μM WP1130 for 2 hours before proteins derived from the total cell lysate (T) and detergent-soluble (S) and insoluble (I) lysates were probed for Bcr-Abl and other proteins or phosphoproteins as indicated. Total cell lysate represents the detergent-soluble and -insoluble lysates combined. Actin served as a protein-loading control. (D) BaF3 cells transformed with eGFP-Bcr-Abl with the T315I mutation were treated with 5μM WP1130 for the intervals noted before detergent-soluble cell lysates were immunoblotted for the indicated protein or phosphoprotein. Actin was blotted as a protein-loading control. Loss of Bcr-Abl from the soluble (cytoplasmic) cell fraction was associated with reduced signaling (pY-Stat5) and the onset of apoptosis (cleaved PARP). (E) Mononuclear cells from 2 CML patients who were progressing on imatinib therapy were treated with 5μM WP1130 for 4 hours before cell lysates representing the combined detergent-soluble and -insoluble fractions were probed for Bcr-Abl by immunoblotting (top). The detergent-soluble cell lysate was also immunoblotted for Bcr-Abl substrate phosphoproteins (pY-Stat5, pY-CrkL), as well as Stat5 and CrkL total protein levels. (F) CD34+ cells from a healthy donor and 2 imatinib-refractory CML patients were treated with 5μM WP1130 for 4 hours before total protein lysates (8 μg) were subjected to ubiquitin immunoblotting. Hsp90 was immunoblotted as a protein-loading control. (G) CD34+ cells isolated from 2 healthy donors and 3 imatinib-refractory CML patients were treated with the indicated concentrations of WP1130 for 24 hours before analysis of cell survival by annexin/propidium iodide staining and flow cytometry. The results represent the average ± SD of 3 replicates. Statistical significance was determined with a paired Student t test.$

WP1130 induces cellular trafficking of Bcr-Abl and inhibits its substrate phosphorylation in CML cells. (A) BV-173 cells were treated with 5μM WP1130 for the intervals indicated before assessing Bcr-Abl and other protein levels in total cell lysates and in the detergent-soluble and -insoluble fractions. Actin was blotted as a protein-loading control. (B) BV-173 and BV-173R cells were treated with 5μM WP1130 for 2 hours before detergent-soluble and -insoluble cell lysates were blotted for Bcr-Abl (K12 antibody) and actin as a protein-loading control. (C) K562R cells were treated with 5μM WP1130 for 2 hours before proteins derived from the total cell lysate (T) and detergent-soluble (S) and insoluble (I) lysates were probed for Bcr-Abl and other proteins or phosphoproteins as indicated. Total cell lysate represents the detergent-soluble and -insoluble lysates combined. Actin served as a protein-loading control. (D) BaF3 cells transformed with eGFP-Bcr-Abl with the T315I mutation were treated with 5μM WP1130 for the intervals noted before detergent-soluble cell lysates were immunoblotted for the indicated protein or phosphoprotein. Actin was blotted as a protein-loading control. Loss of Bcr-Abl from the soluble (cytoplasmic) cell fraction was associated with reduced signaling (pY-Stat5) and the onset of apoptosis (cleaved PARP). (E) Mononuclear cells from 2 CML patients who were progressing on imatinib therapy were treated with 5μM WP1130 for 4 hours before cell lysates representing the combined detergent-soluble and -insoluble fractions were probed for Bcr-Abl by immunoblotting (top). The detergent-soluble cell lysate was also immunoblotted for Bcr-Abl substrate phosphoproteins (pY-Stat5, pY-CrkL), as well as Stat5 and CrkL total protein levels. (F) CD34⁺ cells from a healthy donor and 2 imatinib-refractory CML patients were treated with 5μM WP1130 for 4 hours before total protein lysates (8 μg) were subjected to ubiquitin immunoblotting. Hsp90 was immunoblotted as a protein-loading control. (G) CD34⁺ cells isolated from 2 healthy donors and 3 imatinib-refractory CML patients were treated with the indicated concentrations of WP1130 for 24 hours before analysis of cell survival by annexin/propidium iodide staining and flow cytometry. The results represent the average ± SD of 3 replicates. Statistical significance was determined with a paired Student t test.

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