Vectors used
The MGIFMNOo, MSCV-based retroviral bicistronic construct, contained the Enhanced Green Fluorescent Protein-Internal Ribosomal Entry Site (EGFP-IRES) coding cassette [14] in MGIFMNOo, followed by MPL dimerization inducible construct coding for cytoplasmic domain of mouse MPL linked at its amino end to a 14-amino acid cytoplasmic membrane targeting myristylation domain and at its carboxy end to HA epitope tag. The MGIFMNOo construct contained also sequences coding for the Neomycin resistance gene and the p15 bacterial origin of replication, in its retroviral 3′ untraslated region creating the shuttle plasmid for genomic integration site rescue. The vector was provided by C. Anthony Blau, University of Washington.
The MFhuMIGNOo vector was cloned by replacing the sequences coding for cytoplasmic domain of mouse MPL in MFMIG vector (provided by C. Anthony Blau) with sequences coding for the cytoplasmic domain of human MPL, derived from pNF2hMpl (provided by C. Anthony Blau). The MFhuMIGNOo vector contains sequences coding for dimerization inducible construct based on human MPL upstream of IRES and coding sequences for the EGFP downstream of IRES.
The pEGFP-DLGAP1 vector was cloned by in frame ligation of full length DLGAP1 cDNA into Eco RI and Kpn I sites of the pEGFP-C1 vector (Clontech, Mountain View, CA). The full length DLGAP1 cDNA sequence with 5′ Eco RI site and 3′ Kpn I site was generated on 3197 bp full length cDNA sequence template of DLGAP1 from clone ID: 9020442 (MGC:168065 IMAGE:9020442) in pCR4-TOPO vector, acquired from Open Biosystems (Lafayette, CO). The PCR was performed using Phusion Polymerase system (New England Biolabs, Inc., Ipswich, MA) and primers as follows: forward primer: gcGAATTCcatgaaagggctatcaggc, reverse primer: gaGGTACCctgcgaggtggacaactaca.
The pEGFP-TrDLGAP1 vector was cloned by generating a 5′ end truncated cDNA sequence of DLGAP1 by using forward primer: gcGAATTCccaggatgcctacATGga instead and following the procedure of the pEGFP-DLGAP1 cloning as described above. The forward primer for pEGFP-TrDLGAP1 cloning carries sequence complementary to exon 5 region of DLGAP1 surrounding the first Methionine codon in this exon.
The constitutively active clone of CDK1: Flag-CDK1-AF, and the dominant negative clone of CDK1: Flag-CDK1-ND were a kind gift from Dr. Daniel Wu, VA Puget Sound Health Care System, Seattle.
Cell cultures
Cell lines used include: 293 T [15], K562 (CCL-243, ATCC, Manassas, VA), UT7 [16], UT7/TPO [17], MO7e [18] and HEL [19]. The 293 T cells were grown in Dulbecco’s modified Eagle medium (DMEM (BioWhittaker, Walkersville, MD) with high glucose (4.5 g/liter) supplemented with 10% fetal bovine serum (FBS) (HyClone/Thermo Fisher Scientific Inc., Waltham, MA), with 50 U/ml penicillin and 50 mg/ml streptomycin. The K562 and HEL cell lines were grown in RPMI 1640 (HyClone/Thermo Fisher Scientific Inc., Waltham, MA) supplemented with 10% FBS (HyClone/Thermo Fisher Scientific Inc., Waltham, MA), with 50 U/ml penicillin and 50 mg/ml streptomycin. The UT7 and UT7/TPO were grown in IMDM medium (HyClone/Thermo Fisher Scientific Inc., Waltham, MA) supplemented with 10%, FBS (HyClone/Thermo Fisher Scientific Inc., Waltham, MA), with 50 U/ml penicillin and 50 mg/ml streptomycin. All cell lines were grown at 37 °C in an atmosphere containing 5% CO2.
When indicated, culture medium was supplemented with one of the following reagents: 10 pg/ml AP20187 - a dimerizer of proteins containing an F36 V domain, which is a modified FKBP-binding domain [33] (ARIAD Pharmaceuticals, Cambridge, MA), 1 μM Imatinib (Novartis, Basel, Switzerland), 10 pg/ml GMCSF, 50 ng/ml PEG-rhMGDF, 400 mg/ml Geneticin (Invitrogen, Carlsbad, CA), 50 μM AG490 (Cayman Chemical Co., Ann Arbor, MI [Cayman]), 2.5 μM SU6656 (Cayman), 20 μM U0126 (Cell Signaling Technology, Boston, MA), 100 ng/ml Nocodazole (SCBT), 40 nM PMA (SCBT), 200 μM GTP (SCBT), 50 μM Olomoucine (SCBT), 50 μM iso-Olomoucine (SCBT) and 4.5 μM or 9 μM RO3306 (SCBT).
Exposure times to the different reagents are indicated in the Results section.
Viral transduction
Retroviral supernatants were generated by transient transfection of the packaging 293 T Phoenix gag-pol cells using Polyethylenimine, 25 kDa (Polysciences, Inc., Warrington, PA). Culture supernatants, containing packaged retroviral particles, were mixed with polybrene reagent (8 mg/ml) and used for infection of K562, UT7 and UT7/TPO cells. Cultures were exposed to viral supernatant for 4 h and then received fresh growth medium for 48 h, after that time period transduction efficiency was assayed and cells were supplied with drugs for appropriate selection.
Flow cytometry
Cells were harvested and resuspended in PBS supplemented with 1% FBS. The EGFP expression was analyzed on either a FACSCAN or FACSCalibur (BD Biosciences, San Jose, CA, USA) using argon-ion laser excitation (488 nm). EGFP was detected using the FL1 parameter with emission filter: 530 ± 15 nm. Dead cells and debris were excluded by gating for intact cells using the forward and sideward scatter. Cells non expressing EGFP were used as control to detect autofluorescence. Data acquisition was carried out by analyzing minimum of 10 000 events/sample using CellQuest Software (BD Biosciences).
Cloning and sequencing of retroviral integration sites
Genomic DNA was purified using QiaAmp DNA blood Mini Kit and QiaAmp DNA blood Midi Kit (QiaGen Inc., Valencia, CA) according to manufacturer manuals. The genomic DNA was cut with one of the following enzymes: Eco RI, Bam I, Hind III, Bgl II or Apo I, and liagted with T4 Ligase (New England Biolabs, Ipswich, MA) to recircularize the shuttle plasmids containing fragments of genomic DNA. In following steps the DNA was cut with Dpn I enzyme and elctroporated into DH10B cells. The shuttle plasmids with genomic fragments were purified from single bacterial clones using QiaGen Miniprep Kit and sequenced on ABI-PRISM sequencer using BigDye 3.1 chemistry (Applied Biosystmes, Inc., Foster City, CA) using an oligo primer derived from the sequence of MSCV LTR: 5′-GTTCGCTTCTCGCTTCTGTT-3′.
Immunofluorescent microscopy
For immunofluorescent microscopy studies cells were fixed by adding 1.5 vol. of 3.7% formaldehyde in PBS at room temperature directly to cell culture for 12 min and to transferred to cytospin chambers fitted with Permafrost Plus slides (Fisher Sci., Pittsburgh, PA) and were spinned at 1600 RPM for 2 min in StatSpin Cytofuge (Iris Sample Processing, Westwood, MA). Cells on slides in cytospin chambers were then washed twice in 800 μl PBS and air dried for 16-24 h. For staining cells were permeabilized in 0.3% Triton X-100 in PBS for 10 min at room temperature following with blocking with 5% of appropriate normal serum and 1% BSA (Sigma, Fraction V, lipid-free) in PBS for 1 h at RT. Staining was performed by incubation with desired primary antibody (ies) in PBS containing 1% of appropriate normal serum and 1% BSA for 1 h at RT followed by O/N at 4C. After that cells were washed four times in PBS + 0.05% Tween 20. Incubation with secondary antibodies in PBS containing 1% of appropriate normal serum and 1% BSA was carried for 1.5 h at RT.
The secondary antibodies used were the following: 488 Alexa Fluor rabbit anti-Goat, 488 Alexa Fluor chicken anti-rabbit, 488 Alexa Fluor chicken anti-mouse, 568 Alexa Fluor goat anti-rabbit, 568 Alexa Fluor goat anti-mouse and 568 Alexa Fluor rabbit anti-goat (all from Invitrogen, Grand Island, NY). Cellular DNA was counterstained with 1 μM To-Pro3 Iodide, or 4′6-diamidino-2-phenylindole-2HCl (DAPI) for 15 min at RT, and cells were washed four times in PBS + 0.05% Tween 20. Finally, slides were air dried and coverslips were mounted using one drop of VectaShield solution (Vector Laboratories, Inc., Burlingame, CA).
All imaging was performed on Leica TCS-SP 1 Confocal Laser Scanning System attached to the Leica DM-R Upright Fluorescent microscope (Leica Microsystems Inc., Heidelberg, Germany) or on Nikon Eclipse E-80 microscope fitted with Filters for: DAPI, FITC, TRITC, Cy5 and a camera: QImaging Retiga 2000R (original magnification × 400).
Antibodies
Primary antibodies used in Immunostaining and Western blots included: anti-APC (sc-896, Santa Cruz Biotechnology, Santa Cruz CA [SCBT]), anti-DLGAP1 (sc-25662, SCBT), anti-DLGAP1 (sc-12219, SCBT), anti-DLGAP1 (75–236, NeuroMab/ Antibodies Inc., Davis, CA), anti-PCM-1 (CS #5213, Cell Signaling Technology, Boston, MA), anti-α-Tubulin (13–8000 Zymed/Invitogen, San Francisco, CA), anti-γ-Tubulin (sc-17787, SCBT).
Sequence analysis software
DNA and protein sequence analysis were performed using the Vector NTI software (Invitrogen). The nucleic acid and protein sequence data library searches were performed using Internet based: The Basic Local Alignment Search Tool (BLAST) (National Center for Biotechnology Information) and the University of California Santa Cruz Genome Browser search engines.