Supplementary information

Materials and methods

Antibodies and reagents

Mouse monoclonal anti-Abi1, clone 1B9 (used for IB, IF, and IP), was purchased from MBL International (Woburn, MA); rabbit polyclonal anti-Abi1 (used for IB) was from Novus Biologicals; anti-integrin β7, clone H-120, was from Santa Cruz Biotechnology, Inc. (Dallas, TX); anti β-actin, clone AC-15, was from Sigma-Aldrich (St. Louis, MO); anti-p130 Cas, clone 8G4-E8, was purchased from Upstate/EMD Millipore (San Diego, CA); anti-c-Abl, clone 24-21, was from Calbiochem/EMD Millipore (San Diego, CA). Anti-phospho p130cas, anti-phospho-c-Abl (Tyr412), clone 247C7, anti-p44/42 MAPK (Erk1/2), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), anti-Akt (pan)(11E7), anti-phospho-Akt (Ser473)(D9E), anti-integrin α4, anti-integrin αV, anti-integrin α5, anti-integrin β1, anti-integrin β3, anti-integrin β4, anti-FAK, anti-phospho-FAK (Tyr397), anti-phospho-FAK (Tyr576/577), anti-phospho-FAK (Tyr925), anti-PAK1, anti-PAK2, anti-phospho-PAK1 (Ser144)/PAK2 (Ser141), anti-Src, anti-phospho-Src (Tyr416), anti-CrkL, anti-phospho-CrkL (Tyr207), anti-ILK, and anti-Phospho ILK (Ser246) were from Cell Signaling Technology, Inc. (Danvers, MA). HRP-conjugated goat anti-rabbit IgG (H+L), HRP-conjugated goat anti-mouse IgG (H+L), HRP-conjugated goat Anti-Mouse IgG (Light Chain, used for IP), HRP-conjugated mouse anti-rabbit IgG (Light Chain, used for IP), and mouse IgG were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Alexa Fluor 488 goat anti-mouse secondary antibodies were from Life Technologies (Grand Island, NY). For standard PCR reactions ReadyMix Taq PCR Reaction Mix with MgCl2 was used (Sigma-Aldrich). ABI1 or ITGA4 TaqMan® Gene Expression Assays, human ACTB TaqMan® Pre-Developed Assay Reagent, and TaqMan® Gene Expression Master Mix were purchased from Life Technologies (Grand Island, NY). For CD34+ enrichment, CD34 MicroBead Kit and MACS® Separation LS Columns (Miltenyi Biotec Inc., Auburn, CA) were used. Mounting medium Vectashield with DAPI was purchased from Vector Laboratories, Inc. (Burlingame, CA). Anti-mouse CD49d/VLA-4-FITC was from Southern Biotech (Birmingham, AL). Anti-human CD49d/VLA-4-APC was purchased from BioLegend (San Diego, CA).

Quantitative transcription analysis with real time polymerase chain reaction (qRT-PCR)

Total mRNA was purified using PureLink RNA Mini Kit from Ambion/Life Technologies according to the manufacturer’s protocol. SuperScript First-Strand Synthesis System (Life Technologies) was used for reverse transcription according to the manufacturer’s protocol, and relative levels of RNA were measured by quantitative real-time PCR using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). ABI1, ITGA4, and ACTB were amplified using either SYBR Green Master Mix and gene specific primers or taqman Universal Master Mix (Applied Biosystems) and gene specific TaqMan probes (for details see Supplementary Tables 1 and 2, and Suppl. Fig. 7). Cycle threshold (ct) values of individual genes were subtracted from Ct values for actin (ΔCt), and were then used to calculate fold change in relative gene expression (2–ΔΔCT). For standard PCR reactions, ReadyMix Taq PCR Reaction Mix with MgCl2 (Sigma-Aldrich) was used. Gene expression analysis for cyclin, cyclin kinases, and inhibitors were performed using PrimePCR Custom Plate 96 Well and iTaqTM Universal SYBR® Green Supermix (Bio-Rad). PrimePCR Assay reactions were performed on a C1000TM Thermal Cycler with CFX96TM Real-Time System using the BioRad gene expression assay protocol. Results were evaluated using Bio-Rad CFX manager. Human miR-181a, miR-181a-2*, RNU19, and RNU6B were amplified using TaqMan Universal PCR Master Mix (Applied Biosystems by Life Technologies) and miR specific TaqMan probes (Supplementary Table 2). Relative quantitation of gene expression was evaluated by real-time PCR using two independent systems: CFX96 TM Real-Time System (Bio-Rad) and ViiA™ 7 Real-Time PCR System (Applied Biosystems/Life Technologies).

Immunoblotting

Cells were lysed in LDS Sample Buffer (Life Technologies), reduced for 5 minutes, frozen for 2 h, thawed, and incubated in the presence of Benzonase Nuclease (Novagene/EMD Millipore, San Diego, CA). Samples were sonicated for 18 minutes followed by a final 5 min reduction at 70°C and then evaluated by Western Blotting. Equal loading was ensured by lysing the same number of cells, and was further determined by Coomassie blue staining. After transfer, membranes were blocked for 2 h at RT or overnight at +4°C in 5% milk or 3% BSA (phospho-signal) in TBST buffer. Membranes were incubated with primary antibodies in 2% milk or BSA for 2 h, washed (4x), incubated with secondary antibodies for 1 h, and washed (4x). Chemiluminescent signal was obtained using SuperSignal West Pico Substrate (Thermo Scientific, Rockford, IL) and Konica Minolta SRX-101A developer.

Affymetrix miRNA arrays

Small RNA was labeled using the Flash-tag labeling Kit from Affymetrix (Santa Clara, CA). Labeled miRNAs were run on an RNA6000 Bioanalyzer chip to verify equimolar concentration of small RNAs in all samples prior to adding RNA to the hybridization cocktail. Small RNAs were hybridized for 17 h at 48°C, 60 rpm to Affymetrix miRNA v3.0 arrays. Affymetrix Wash/Stain Kit was used to stain hybridized RNA. Non-specifically bound RNA was removed using the Affymetrix FS450 Fluidics Station and wash/stain protocol FS450_0002. RNA was visualized using an Affymetrix 3000 7G scanner. Partek Genomics Suite version 6.6 was used to assess the quality of the arrays and to detect differentially expressed genes. To this end RMA was employed and only genes in the resulting ANOVA file which were at least 2 fold up- or down- regulated and had an unadjusted p-value of ≤ 0.05 were included.

Transfection

K562cII or K562-STI-R cells were transiently transfected with the Abi1 silencing pGFP-V-RS vector encoding a 29 mer shRNA (OriGene Technologies, Inc.) or Abi1-YFP (a kind gift from Dr. Ann M. Pendergast). Lipofectamine 2000 was used as a transfecting agent. Transfection efficiency was determined to be 20-25% for both Abi1 overexpressing or silencing vectors.

Engraftment of human cells in immunodeficient mice

K562 cells were transfected with vector encoding YFP tagged Abi1. Lipofectamine 2000 was used for the experiment (Life Technologies) in accordance to manufacturer’s instructions. Transfection efficiency was 24%. Control (K562cI) or Abi1 overexpressing cells (K562cI/Abi1) were harvested and transplanted via tail vein injection (2x106 cells/mouse) into 12 week old sub-lethally irradiated (3.8 Gy) NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Jackson Laboratory, Bar Harbor, ME, stock#005557). Mice were euthanized after 4 weeks, and marrow contents of femurs as well as spleens, livers, and tumors were obtained and subjected to pathological evaluation. Leukemic cell engraftment was assessed by FISH. BCR-ABL protein levels in marrow cells obtained from mice were evaluated by immunoblotting.

Confocal microscopy

Cytospins were prepared in a Cytospin centrifuge (Shandon Cytospin 3). Cells were fixed for 20 min in 4% paraformaldehyde in PBS pH 7.4, permeabilized with 4% PFA solution containing 0.1% Triton X-100 for 1 minute, washed with PBS, blocked for 1 h in PBS containing 10% FBS, followed by incubation with primary antibodies for 1 h and secondary antibodies for 0.5 h. Slides were mounted using mounting medium with DAPI (Vector Laboratories). Images were acquired with a Zeiss LSM700, 63x/1.4 oil Plan Apochromat objective.

Immunoprecipitation

Cells were incubated for 10 min in ice cold RIPA buffer supplemented with 10 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, and protease inhibitor cocktail (Millipore). Lysates were clarified by centrifugation at 21,000 x g 10min at 4°C. Protein concentration was adjusted to 1 mg/ml in all samples, which were then incubated for 2 h in the presence of 5 mg/ml IgG and protein G agarose beads (Thermo Scientific). Next, the pre-cleared samples were incubated overnight at 4°C in the presence of primary antibody (5µg per 1 mg of total protein) followed by incubation with protein G agarose beads for 2 h at 4°C. Beads were then washed 3x in RIPA buffer with inhibitors, reduced in the presence of LDS Sample Buffer, and analyzed by immunoblotting.

Flow Cytometric Analysis

1×106 cells were washed briefly in PBS, incubated for 1 h with anti-mouse CD49d/VLA-4-FITC or anti-human CD49d/VLA-4-APC or their isotype controls at 4°C in the dark, washed 3x in PBS, and analyzed on an LSRII flow cytometer (Becton Dickinson).

Proliferation, adhesion, and gene expression in the presence of VCAM-1.

For proliferation, adhesion, and gene expression experiments, due to the short half-life of VCAM-1 at 37°C, cells were re-plated on fresh VCAM-1-coated wells every 24 h. For proliferation assays, 24-well or 6-well plates were coated with 5 µg/mL VCAM-1 in PBS overnight at 4°C. Cells were seeded at a density of 2.5×104 or 1x106 cells per well in triplicate wells. Cells were counted after 96 h incubation. For attachment assays, cells were seeded on VCAM-1 coated plates and allowed to attach for 8h. Plates were shaken and movement was recorded on an Axiovert 200M (Zeiss) microscope equipped with a CCD camera (Hammamatsu) using a 20x/0.4 LD Achroplan objective. For assaying expression of cyclins, cyclin kinases, and inhibitors, cells were seeded on VCAM-1 coated plates and harvested and mRNA was isolated after 96 h.

Colony forming assay

Cells (5x103) were suspended in methylcellulose (Methocult, StemCell Technologies Inc., Vancouver, Canada) supplemented with 10% FBS and 2 mM Glutamax and plated on 6 cm plates in triplicate. The plates were incubated at 37°C, 5% CO2, and colony number was evaluated after 14 days. Images were taken with a Zeiss Axiovert 200M using a 20x/0.4 LD Achroplan objective.

Attachment evaluation

K562 control (cII) or K562-STI-R resistant cells were seeded on 6-well plates with (i) standard negatively charged hydrophilic surface (treated polystyrene), (ii) uncharged hydrophobic surface (untreated polystyrene), or (iii) neutral hydrophilic hydrogel coated surface (ultra-low attachment polystyrene) (Corning). Cells were grown for 12 days with regular passages. Cells were removed on day 12, and plates were gently washed with pre-warmed PBS (3x). Photomicrographs were obtained before and after wash using a Zeiss Observer Z1 inverted microscope equipped with an AxioCamHRc color camera. A Plan-APOCHROMAT 10x/0,45 Ph1 objective was used.

Statistical Analysis

Data obtained were from experiments performed in biological triplicates or quadruplicates in three experimental sets. Quantitative data are shown as mean ± standard deviation (SD). Statistical significance was determined using the unpaired Student-t test. A value of p<0.05 was considered to be significant.

Supplementary figure legends

Suppl. Fig. 1. Images presenting K562 cII (A) or K562-STI-R cells (B) growing on standard cell culture treated, untreated, or ultra-low attachment polystyrene plates. Images were taken before and after washing with warm PBS. (C) Quantification of cells growing on standard cell culture treated, untreated, or ultra-low attachment polystyrene plates indicated that 70%, 47% and 21% of K562-STI-R cells respectively, remained attached to the plates after the PBS wash. No attachment for K562 control cells (cII) after PBS wash was observed.

Suppl. Fig. 2. IM resistant cells acquire an anchorage dependent phenotype. Colony formation assay was performed using Methocult semi-solid media. The number of colonies was evaluated 14 days after initial plating of cells. IM was present in semi-solid media at 0.1 µM for K562-STI-S cells and 0.6 µM for K562-STI-R and Ba/F3 mutants. Images presenting colony formation by K562 (A) and Ba/F3 cells (B) in semi-solid media. Insets show image of the culture plate. A nearly 50% decrease in colony formation was observed for K562-STI-S cells (C) and more than 90% decrease was observed for K562-STI-R cells (C). Some decrease in colony formation was observed also for cells expressing Bcr-Abl mutants (D), with Ba/F3 M351T exhibiting the most significant decrease in the number of colonies per cm2. All images were acquired using a 5x objective on a Zeiss Axioplan microscope equipped with a CCD camera. All analyzed cell lines were plated in triplicate, and the experiment was performed three times.

Suppl. Fig. 3. Histograms presenting isotype controls for the expression of CD49d (α4 integrin) in K562 (A) and Ba/F3 cells (B) as presented in Fig. 3. G, H.

Suppl. Fig. 4. Expression of NANOG, POU5F1 and SOX2 was evaluated by qPCR in K562-STI-R vs. K562 ctrl II cells cultured for 96h over the VCAM-1 coated surfaces. More than 3-fold upregulation of NANOG and SOX2 was observed in K562-STI-R cells either grown on uncoated (A) or VCAM-1 coated surfaces (B).

Suppl. Fig. 5. Abi1 associates with α4 and Bcr-Abl. Immunoprecipitation experiments using anti-Abi1 antibody (clone 1B9) showed that Abi1 associates with both α4 integrin and Bcr-Abl in K562 (A) and Ba/F3 (B) cells. IP was performed in RIPA buffer supplemented with phosphatase and proteases inhibitors. An association between Abi1 and the full length 150 kDa fragment of α4 integrin was noted only in K562-STI-R (A) cells. In K562-STI-S (A) and Ba/F3 (B) cells an association between Abi1 and the 70/80 kDa fragment of α4 was detected. Absence of a detectable Bcr-Abl band in the K562-STI-R immunoprecipitate was due to the initial low levels of Bcr-Abl in these cells (A). Immunoprecipitation of α4 integrin and Bcr-Abl in LAMA-84 control (-S) and IM resistant (-R) cells growing in 0.6 µM IM, showed that Abi1 co-immunoprecipitates with α4 150 and 70/80 kDa fragments and with Bcr-Abl (C). IP was performed independently three times; representative results are presented.

Suppl. Fig. 6. Activation of Integrin Linked Kinase (ILK). Because α4 integrin forms dimers with the β1 subunit, and signaling cascades may be activated via activation of β1 integrin, we evaluated the status of activation of the immediate β1 activation effector – Integrin Linked Kinase (ILK). Western blot evaluation demonstrated lack of activation of ILK seen as a lack of phospho-signal on Ser246 of ILK in K562-STI-S and –R cells (A). Activity of ILK was at a relatively low level and unchanged in all Ba/F3 cells (B). Absence of Abi1 affects both α4 and Bcr-Abl signaling in IM resistant cells. Western blot evaluation of the activation status of the several downstream effectors of α4 signaling cascade revealed phosphorylation of FAK (on Tyr397, 576/577, 925), Src (Tyr416), p130 Cas (Tyr762), and PAK (Ser144/141) in K562-STI-S cells (C). In K562-STI-R cells, we noted decreased phosphorylation of FAK, Src, p130 Cas, and PAK (C). FAK, Src, p130 Cas, and PAK remained relatively unchanged, demonstrating a high steady state level of activation in all analyzed Ba/F3 cells (D). Representative data from three independent experiments are presented.

Suppl. Fig. 7. PCR confirmation of correct amplicon sizes generated by designed primer pairs to evaluate ABI1, ITGA4, and ACTB transcript levels using SYBR green based RT-qPCR for K562 and Ba/F3 cells. For amplicon sizes please refer to Suppl. Table 1 and 2.

Suppl. Movies 1-2. Plates with K562 (SM1) or Ba/F3 cells (SM2) were briefly shaken during their normal growth on plastic in order to register possible attachment of cells to the uncoated surface. K562-STI-R cells displayed more than 50% attachment in comparison to control cells growing in suspension (1SM D, E). Irregular shape with protrusions and no attachment can be seen for K562-STI-S (1SM A, B, C) or Ba/F3 cells (2SM A, B, C, D).

Suppl. Movies 3-4. Cells grown on VCAM-1 coated surfaces (5 μg/mL) were briefly shaken, and cell attachment upon shaking was recorded. Nearly 100% of all analyzed cells except K562 ctrl I and II were found to be attached to VCAM-1 coated surfaces (SM3 A-E, SM4 A-D).

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