Bcr-Abl induces abnormal cytoskeleton remodeling, 1 integrin clustering and increased cell adhesion to fibronectin through the Abl interactor 1 pathway

JCS03430 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Specificity of anti-Abi1 antibody and downregulation of Abi2 in murine hematopoietic cell lines transformed by p185^wt. (A) Myc-tagged Abi1 and Abi2 were produced in vitro by coupled transcription/translation and subjected to western blot analysis using indicated antibodies. (B) Ba/F3 and 32D cells were transduced with empty retroviral vector (control) or retroviral vector expressing p185^wt. Total lysates from 1×10⁶ cells were analyzed by western blot using anti-Abi2 antibody.

• Supplemental Figure 2 -

  Fig. S2. PI3K pathway is not required for Bcr-Abl-induced abnormal F-actin remodeling. (A) Imatinib, but not LY294002, inhibited Bcr-Abl-induced abnormal actin cytoskeleton remodeling. Ba/F3 cells (a) and the Ba/F3 transformed by p185^wt (b) were treated without (a,b) or with either 1 μM imatinib (c) or 50 μM LY294002 (d). The cells were fixed, permeablized and stained with TRITC-conjugated phalloidin and DAPI to visualize F-actin and nuclei, respectively. Images were captured by fluorescence microscope. Arrowheads indicate the abnormal F-actin-enriched structures. (B) Tyrosine 407 is not required for Bcr-Abl-induced membrane translocation of Abi1 and actin remodeling. Ba/F3 cells and the Ba/F3 cells transformed by p185^wt were transduced with retroviruses expressing either GFP-Abi1 or GFP-Abi1^Y407F, as indicated. Cells were fixed, permeablized and stained by TRITC-conjugated phalloidin and DAPI. Images were captured by two-photon confocal microscope. Bar, 10 μm.

• Supplemental Figure 3 -

  Fig. S3. Deletion of the SH3 domain and C-terminal proline-rich sequences in Bcr-Abl abrogates its signal transduction to Abi1. (A) Schematic representation of p185^wt and p185^ΔSH3ΔC. (B) The p185^ΔSH3ΔC is deficient in binding to Abi1 and inducing Abi1 phosphorylation. The lysates from Ba/F3 cells transduced with empty retroviral vector (C) or the retroviral vectors expressing either p185^wt (WT) or p185^ΔSH3ΔC (Mu) were immunoprecipitated with anti-Abi1 antibody. The immunoprecipitates (middle and lower panels) and 1/50 of total cell lysates used for immunoprecipitation (TCL, upper panel) were subjected to western blot analysis using antibodies specific for Abl, Abi1 and phosphotyrosine (pTyr), as indicated. The anti-Abl antibody recognizes both Bcr-Abl and endogenous c-Abl.

• Supplemental Figure 4 -

  Fig. S4. Abi1^PPLL retains the ability to interact with WAVE2 and Hem1. (A) The p185^wt-transformed Ba/F3 cells were transduced with retroviruses expressing either HA-tagged Abi1 or Abi1^PPLL. The parental cells (lane 1) as well as the cells that express HA-Abi1 (lane 2) or HA-Abi1^PPLL (lane 3) were lysed and immunoprecipitated with an antibody specific for the HA-tag. The immunoprecipitates were subjected to western blot (WB) analysis using indicated antibodies. (B) The parental p185^wt-transformed Ba/F3 cells (lane 1) and the p185^wt-transformed cells expressing myc-tagged Hem1 (lanes 2 and 3) were transduced with retroviruses expressing either HA-tagged Abi1 (lane 2) or Abi1^PPLL (lane 3). The cell lysates were immunoprecipitated by anti-HA antibody and the immunoprecipitates were analyzed by western blot using indicated antibodies.

• Supplemental Figure 5 -

  Fig. S5. The β1-integrin protein level is not elevated among Ba/F3 cells and the Ba/F3 cells expressing the wild-type and mutant forms of Bcr-Abl and Abi1. (A) Lysates from Ba/F3 cells and the Ba/F3 cells expressing p185^wt, p185^ΔSH3ΔC, and p185^wt plus Abi1^PPLL, as indicated, were subjected to western blot analysis using anti-β1-integrin antibody. The western blot analysis using anti-β-actin antibody was also included to show equal loading. (B) Analysis of surface β1-integrin level by flow cytometry. Parental Ba/F3 cells and Ba/F3 cells expressing p185^wt, p185^ΔSH3ΔC, or p185^wt plus Abi1^PPLL, as indicated, were fixed and probed with anti-β1-integrin antibody, or normal rabbit IgG as control. Cells were then stained by Alexa-conjugated secondary antibody and subjected to flow cytometry analysis. MFI, median fluorescence intensities.

• Supplemental Figure 6 -

  Fig. S6. β1-integrin is clustered and colocalized with abnormal F-actin-enriched structures in p185^wt-transformed Ba/F3 cells adherent to fibronectin-coated surfaces. The Ba/F3 cells and Ba/F3 cells expressing either p185^wt or p185^ΔSH3ΔC, as indicated, were plated on glass coverslips coated with fibronectin and grown for 16 hours. Adherent cells were fixed, permeablized and probed with rat monoclonal anti-β1-integrin antibody. Cells were then stained with FITC-conjugated mouse anti-rat IgG for β1-integrin (green), TRITC-conjugated phalloidin for F-actin (red), and DAPI for nuclei (blue), as indicated. The images were captured by a Nikon TE-2000 fluorescence microscope. β1-integrin clustering is indicated by arrowheads and the colocalization of integrin with abnormal actin-enriched structure is shown in merged images.

• Supplemental Figure 7 -

  Fig. S7. Expression of Abi1^PPLL inhibited Bcr-Abl-induced spontaneous cell migration on fibronectin-coated surfaces. Ba/F3 cells and the Ba/F3 cells expressing p185^wt were transduced with retrovirus expressing GFP-Abi1^PPLL. Spontaneous cell migration on fibronectin-coated surfaces was determined by Transwell migration assay. The vertical axis shows the percentage of migrated cells and is expressed as the mean ± s.d. of triplicate wells. *P<0.05 versus p185^wt-expressing cells (n=3). Data are representative of three independent experiments.