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Supplementary information
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Materials and methods
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Antibodies and reagents
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Mouse monoclonal anti-Abi1, clone 1B9 (used for IB, IF, and IP), was purchased from MBL
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International (Woburn, MA); rabbit polyclonal anti-Abi1 (used for IB) was from Novus
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Biologicals; anti-integrin β7, clone H-120, was from Santa Cruz Biotechnology, Inc. (Dallas, TX);
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anti β-actin, clone AC-15, was from Sigma-Aldrich (St. Louis, MO); anti-p130 Cas, clone 8G4-E8,
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was purchased from Upstate/EMD Millipore (San Diego, CA); anti-c-Abl, clone 24-21, was from
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Calbiochem/EMD Millipore (San Diego, CA). Anti-phospho p130cas, anti-phospho-c-Abl
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(Tyr412), clone 247C7, anti-p44/42 MAPK (Erk1/2), anti-phospho-p44/42 MAPK (Erk1/2)
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(Thr202/Tyr204), anti-Akt (pan)(11E7), anti-phospho-Akt (Ser473)(D9E), anti-integrin α4, anti-
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integrin αV, anti-integrin α5, anti-integrin β1, anti-integrin β3, anti-integrin β4, anti-FAK, anti-
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phospho-FAK (Tyr397), anti-phospho-FAK (Tyr576/577), anti-phospho-FAK (Tyr925), anti-PAK1,
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anti-PAK2, anti-phospho-PAK1 (Ser144)/PAK2 (Ser141), anti-Src, anti-phospho-Src (Tyr416),
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anti-CrkL, anti-phospho-CrkL (Tyr207), anti-ILK, and anti-Phospho ILK (Ser246) were from Cell
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Signaling Technology, Inc. (Danvers, MA). HRP-conjugated goat anti-rabbit IgG (H+L), HRP-
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conjugated goat anti-mouse IgG (H+L), HRP-conjugated goat Anti-Mouse IgG (Light Chain, used
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for IP), HRP-conjugated mouse anti-rabbit IgG (Light Chain, used for IP), and mouse IgG were
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from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Alexa Fluor 488 goat anti-
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mouse secondary antibodies were from Life Technologies (Grand Island, NY). For standard PCR
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reactions ReadyMix Taq PCR Reaction Mix with MgCl2 was used (Sigma-Aldrich). ABI1 or ITGA4
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TaqMan® Gene Expression Assays, human ACTB TaqMan® Pre-Developed Assay Reagent, and
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TaqMan® Gene Expression Master Mix were purchased from Life Technologies (Grand Island,
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NY). For CD34+ enrichment, CD34 MicroBead Kit and MACS® Separation LS Columns (Miltenyi
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Biotec Inc., Auburn, CA) were used. Mounting medium Vectashield with DAPI was purchased
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from Vector Laboratories, Inc. (Burlingame, CA). Anti-mouse CD49d/VLA-4-FITC was from
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Southern Biotech (Birmingham, AL). Anti-human CD49d/VLA-4-APC was purchased from
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BioLegend (San Diego, CA).
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Quantitative transcription analysis with real time polymerase chain reaction (qRT-PCR)
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Total mRNA was purified using PureLink RNA Mini Kit from Ambion/Life Technologies according
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to the manufacturer’s protocol. SuperScript First-Strand Synthesis System (Life Technologies)
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was used for reverse transcription according to the manufacturer’s protocol, and relative levels
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of RNA were measured by quantitative real-time PCR using the ABI PRISM 7000 Sequence
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Detection System (Applied Biosystems, Foster City, CA). ABI1, ITGA4, and ACTB were amplified
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using either SYBR Green Master Mix and gene specific primers or taqman Universal Master Mix
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(Applied Biosystems) and gene specific TaqMan probes (for details see Supplementary Tables 1
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and 2, and Suppl. Fig. 7). Cycle threshold (ct) values of individual genes were subtracted from Ct
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values for actin (ΔCt), and were then used to calculate fold change in relative gene expression
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(2–ΔΔCT). For standard PCR reactions, ReadyMix Taq PCR Reaction Mix with MgCl 2 (Sigma-
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Aldrich) was used. Gene expression analysis for cyclin, cyclin kinases, and inhibitors were
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performed using PrimePCR Custom Plate 96 Well and iTaqTM Universal SYBR® Green Supermix
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(Bio-Rad). PrimePCR Assay reactions were performed on a C1000TM Thermal Cycler with
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CFX96TM Real-Time System using the BioRad gene expression assay protocol. Results were
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evaluated using Bio-Rad CFX manager. Human miR-181a, miR-181a-2*, RNU19, and RNU6B
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were amplified using TaqMan Universal PCR Master Mix (Applied Biosystems by Life
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Technologies) and miR specific TaqMan probes (Supplementary Table 2). Relative quantitation
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of gene expression was evaluated by real-time PCR using two independent systems: CFX96 TM
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Real-Time System (Bio-Rad) and ViiA™ 7 Real-Time PCR System (Applied Biosystems/Life
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Technologies).
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Immunoblotting
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Cells were lysed in LDS Sample Buffer (Life Technologies), reduced for 5 minutes, frozen for 2 h,
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thawed, and incubated in the presence of Benzonase Nuclease (Novagene/EMD Millipore, San
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Diego, CA). Samples were sonicated for 18 minutes followed by a final 5 min reduction at 70°C
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and then evaluated by Western Blotting. Equal loading was ensured by lysing the same number
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of cells, and was further determined by Coomassie blue staining. After transfer, membranes
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were blocked for 2 h at RT or overnight at +4°C in 5% milk or 3% BSA (phospho-signal) in TBST
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buffer. Membranes were incubated with primary antibodies in 2% milk or BSA for 2 h, washed
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(4x), incubated with secondary antibodies for 1 h, and washed (4x). Chemiluminescent signal
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was obtained using SuperSignal West Pico Substrate (Thermo Scientific, Rockford, IL) and
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Konica Minolta SRX-101A developer.
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Affymetrix miRNA arrays
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Small RNA was labeled using the Flash-tag labeling Kit from Affymetrix (Santa Clara, CA).
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Labeled miRNAs were run on an RNA6000 Bioanalyzer chip to verify equimolar concentration of
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small RNAs in all samples prior to adding RNA to the hybridization cocktail. Small RNAs were
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hybridized for 17 h at 48°C, 60 rpm to Affymetrix miRNA v3.0 arrays. Affymetrix Wash/Stain Kit
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was used to stain hybridized RNA. Non-specifically bound RNA was removed using the
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Affymetrix FS450 Fluidics Station and wash/stain protocol FS450_0002. RNA was visualized
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using an Affymetrix 3000 7G scanner. Partek Genomics Suite version 6.6 was used to assess the
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quality of the arrays and to detect differentially expressed genes. To this end RMA was
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employed and only genes in the resulting ANOVA file which were at least 2 fold up- or down-
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regulated and had an unadjusted p-value of ≤ 0.05 were included.
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Transfection
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K562cII or K562-STI-R cells were transiently transfected with the Abi1 silencing pGFP-V-RS
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vector encoding a 29 mer shRNA (OriGene Technologies, Inc.) or Abi1-YFP (a kind gift from Dr.
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Ann M. Pendergast). Lipofectamine 2000 was used as a transfecting agent. Transfection
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efficiency was determined to be 20-25% for both Abi1 overexpressing or silencing vectors.
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Engraftment of human cells in immunodeficient mice
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K562 cells were transfected with vector encoding YFP tagged Abi1. Lipofectamine 2000 was
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used for the experiment (Life Technologies) in accordance to manufacturer’s instructions.
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Transfection efficiency was 24%. Control (K562cI) or Abi1 overexpressing cells (K562cI/Abi1)
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were harvested and transplanted via tail vein injection (2x106 cells/mouse) into 12 week old
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sub-lethally irradiated (3.8 Gy) NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Jackson Laboratory,
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Bar Harbor, ME, stock#005557). Mice were euthanized after 4 weeks, and marrow contents of
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femurs as well as spleens, livers, and tumors were obtained and subjected to pathological
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evaluation. Leukemic cell engraftment was assessed by FISH. BCR-ABL protein levels in marrow
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cells obtained from mice were evaluated by immunoblotting.
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Confocal microscopy
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Cytospins were prepared in a Cytospin centrifuge (Shandon Cytospin 3). Cells were fixed for 20
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min in 4% paraformaldehyde in PBS pH 7.4, permeabilized with 4% PFA solution containing
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0.1% Triton X-100 for 1 minute, washed with PBS, blocked for 1 h in PBS containing 10% FBS,
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followed by incubation with primary antibodies for 1 h and secondary antibodies for 0.5 h.
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Slides were mounted using mounting medium with DAPI (Vector Laboratories). Images were
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acquired with a Zeiss LSM700, 63x/1.4 oil Plan Apochromat objective.
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Immunoprecipitation
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Cells were incubated for 10 min in ice cold RIPA buffer supplemented with 10 mM sodium
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fluoride, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, and protease inhibitor
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cocktail (Millipore). Lysates were clarified by centrifugation at 21,000 x g 10min at 4°C. Protein
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concentration was adjusted to 1 mg/ml in all samples, which were then incubated for 2 h in the
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presence of 5 mg/ml IgG and protein G agarose beads (Thermo Scientific). Next, the pre-cleared
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samples were incubated overnight at 4°C in the presence of primary antibody (5µg per 1 mg of
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total protein) followed by incubation with protein G agarose beads for 2 h at 4°C. Beads were
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then washed 3x in RIPA buffer with inhibitors, reduced in the presence of LDS Sample Buffer,
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and analyzed by immunoblotting.
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Flow Cytometric Analysis
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1×106 cells were washed briefly in PBS, incubated for 1 h with anti-mouse CD49d/VLA-4-FITC or
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anti-human CD49d/VLA-4-APC or their isotype controls at 4°C in the dark, washed 3x in PBS,
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and analyzed on an LSRII flow cytometer (Becton Dickinson).
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Proliferation, adhesion, and gene expression in the presence of VCAM-1.
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For proliferation, adhesion, and gene expression experiments, due to the short half-life of
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VCAM-1 at 37°C, cells were re-plated on fresh VCAM-1-coated wells every 24 h. For
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proliferation assays, 24-well or 6-well plates were coated with 5 µg/mL VCAM-1 in PBS
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overnight at 4°C. Cells were seeded at a density of 2.5×104 or 1x106 cells per well in triplicate
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wells. Cells were counted after 96 h incubation. For attachment assays, cells were seeded on
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VCAM-1 coated plates and allowed to attach for 8h. Plates were shaken and movement was
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recorded on an Axiovert 200M (Zeiss) microscope equipped with a CCD camera (Hammamatsu)
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using a 20x/0.4 LD Achroplan objective. For assaying expression of cyclins, cyclin kinases, and
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inhibitors, cells were seeded on VCAM-1 coated plates and harvested and mRNA was isolated
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after 96 h.
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Colony forming assay
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Cells (5x103) were suspended in methylcellulose (Methocult, StemCell Technologies Inc.,
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Vancouver, Canada) supplemented with 10% FBS and 2 mM Glutamax and plated on 6 cm
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plates in triplicate. The plates were incubated at 37°C, 5% CO2, and colony number was
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evaluated after 14 days. Images were taken with a Zeiss Axiovert 200M using a 20x/0.4 LD
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Achroplan objective.
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Attachment evaluation
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K562 control (cII) or K562-STI-R resistant cells were seeded on 6-well plates with (i) standard
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negatively charged hydrophilic surface (treated polystyrene), (ii) uncharged hydrophobic
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surface (untreated polystyrene), or (iii) neutral hydrophilic hydrogel coated surface (ultra-low
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attachment polystyrene) (Corning). Cells were grown for 12 days with regular passages. Cells
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were removed on day 12, and plates were gently washed with pre-warmed PBS (3x).
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Photomicrographs were obtained before and after wash using a Zeiss Observer Z1 inverted
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microscope equipped with an AxioCamHRc color camera. A Plan-APOCHROMAT 10x/0,45 Ph1
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objective was used.
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Statistical Analysis
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Data obtained were from experiments performed in biological triplicates or quadruplicates in
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three experimental sets. Quantitative data are shown as mean ± standard deviation (SD).
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Statistical significance was determined using the unpaired Student-t test. A value of p<0.05 was
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considered to be significant.
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Supplementary figure legends
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Suppl. Fig. 1. Images presenting K562 cII (A) or K562-STI-R cells (B) growing on standard cell
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culture treated, untreated, or ultra-low attachment polystyrene plates. Images were taken
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before and after washing with warm PBS. (C) Quantification of cells growing on standard cell
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culture treated, untreated, or ultra-low attachment polystyrene plates indicated that 70%, 47%
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and 21% of K562-STI-R cells respectively, remained attached to the plates after the PBS wash.
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No attachment for K562 control cells (cII) after PBS wash was observed.
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Suppl. Fig. 2. IM resistant cells acquire an anchorage dependent phenotype. Colony formation
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assay was performed using Methocult semi-solid media. The number of colonies was evaluated
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14 days after initial plating of cells. IM was present in semi-solid media at 0.1 µM for K562-STI-S
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cells and 0.6 µM for K562-STI-R and Ba/F3 mutants. Images presenting colony formation by
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K562 (A) and Ba/F3 cells (B) in semi-solid media. Insets show image of the culture plate. A
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nearly 50% decrease in colony formation was observed for K562-STI-S cells (C) and more than
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90% decrease was observed for K562-STI-R cells (C). Some decrease in colony formation was
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observed also for cells expressing Bcr-Abl mutants (D), with Ba/F3 M351T exhibiting the most
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significant decrease in the number of colonies per cm2. All images were acquired using a 5x
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objective on a Zeiss Axioplan microscope equipped with a CCD camera. All analyzed cell lines
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were plated in triplicate, and the experiment was performed three times.
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Suppl. Fig. 3. Histograms presenting isotype controls for the expression of CD49d (α4 integrin)
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in K562 (A) and Ba/F3 cells (B) as presented in Fig. 3. G, H.
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Suppl. Fig. 4. Expression of NANOG, POU5F1 and SOX2 was evaluated by qPCR in K562-STI-R vs.
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K562 ctrl II cells cultured for 96h over the VCAM-1 coated surfaces. More than 3-fold
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upregulation of NANOG and SOX2 was observed in K562-STI-R cells either grown on uncoated
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(A) or VCAM-1 coated surfaces (B).
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Suppl. Fig. 5. Abi1 associates with α4 and Bcr-Abl. Immunoprecipitation experiments using
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anti-Abi1 antibody (clone 1B9) showed that Abi1 associates with both α4 integrin and Bcr-Abl in
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K562 (A) and Ba/F3 (B) cells. IP was performed in RIPA buffer supplemented with phosphatase
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and proteases inhibitors. An association between Abi1 and the full length 150 kDa fragment of
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α4 integrin was noted only in K562-STI-R (A) cells. In K562-STI-S (A) and Ba/F3 (B) cells an
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association between Abi1 and the 70/80 kDa fragment of α4 was detected. Absence of a
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detectable Bcr-Abl band in the K562-STI-R immunoprecipitate was due to the initial low levels
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of Bcr-Abl in these cells (A). Immunoprecipitation of α4 integrin and Bcr-Abl in LAMA-84 control
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(-S) and IM resistant (-R) cells growing in 0.6 µM IM, showed that Abi1 co-immunoprecipitates
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with α4 150 and 70/80 kDa fragments and with Bcr-Abl (C). IP was performed independently
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three times; representative results are presented.
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Suppl. Fig. 6. Activation of Integrin Linked Kinase (ILK). Because α4 integrin forms dimers with
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the β1 subunit, and signaling cascades may be activated via activation of β1 integrin, we
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evaluated the status of activation of the immediate β1 activation effector – Integrin Linked
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Kinase (ILK). Western blot evaluation demonstrated lack of activation of ILK seen as a lack of
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phospho-signal on Ser246 of ILK in K562-STI-S and –R cells (A). Activity of ILK was at a relatively
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low level and unchanged in all Ba/F3 cells (B). Absence of Abi1 affects both α4 and Bcr-Abl
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signaling in IM resistant cells. Western blot evaluation of the activation status of the several
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downstream effectors of α4 signaling cascade revealed phosphorylation of FAK (on Tyr397,
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576/577, 925), Src (Tyr416), p130 Cas (Tyr762), and PAK (Ser144/141) in K562-STI-S cells (C). In
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K562-STI-R cells, we noted decreased phosphorylation of FAK, Src, p130 Cas, and PAK (C). FAK,
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Src, p130 Cas, and PAK remained relatively unchanged, demonstrating a high steady state level
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of activation in all analyzed Ba/F3 cells (D). Representative data from three independent
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experiments are presented.
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Suppl. Fig. 7. PCR confirmation of correct amplicon sizes generated by designed primer pairs to
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evaluate ABI1, ITGA4, and ACTB transcript levels using SYBR green based RT-qPCR for K562 and
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Ba/F3 cells. For amplicon sizes please refer to Suppl. Table 1 and 2.
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Suppl. Movies 1-2. Plates with K562 (SM1) or Ba/F3 cells (SM2) were briefly shaken during their
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normal growth on plastic in order to register possible attachment of cells to the uncoated
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surface. K562-STI-R cells displayed more than 50% attachment in comparison to control cells
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growing in suspension (1SM D, E). Irregular shape with protrusions and no attachment can be
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seen for K562-STI-S (1SM A, B, C) or Ba/F3 cells (2SM A, B, C, D).
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Suppl. Movies 3-4. Cells grown on VCAM-1 coated surfaces (5 μg/mL) were briefly shaken, and
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cell attachment upon shaking was recorded. Nearly 100% of all analyzed cells except K562 ctrl I
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and II were found to be attached to VCAM-1 coated surfaces (SM3 A-E, SM4 A-D).
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