Transgenic mouse models
Transgenic mice used in this work have been previously described . C3G (full-length) and C3GΔCat (deleted in the catalytic region) transgenes from human origin are expressed under the control of the megakaryocyte and platelet specific PF4 gene promoter. Transgenic C3G (Tg-C3G) lines 2C1 and 6A6 were used. For transgenic C3GΔCat (Tg-C3GΔCat), line 8A3 was used. All mice used in these studies were 8–12 week-old.
Cell lines and Bone Marrow cell cultures
K562 (ATCC, CCL243) and HEL (ATCC TIB-180) leukemic cell lines were maintained in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS), 2 mM Glutamine, 100 U/ml Penicillin and 100 μg/ml Streptomycin (Gibco). HEK-293 T (ATCC, CRL-3216, human embryonic kidney) and B16-F10 (ATCC, CRL-6475, murine melanoma) cell lines were grown in DMEM medium (Sigma) containing 10% FBS, 2 mM Glutamine, 100 U/ml Penicillin and 100 μg/ml Streptomycin. The megakaryocytic cell line, Dami, was grown in IMDM medium (Gibco) supplemented with 10% horse serum (HyClone), 20 mM Hepes, 100 U/ml Penicillin and 100 μg/ml Streptomycin (Gibco). Dami cells overexpressing GATA-1 fusioned to V5 and those expressing the empty lentiviral vector pLenti6 V5/LacZ (Gateway) were also grown in this medium but in the presence of 2 μg/ml blasticidine.
Bone marrow cells (BMCs) were obtained from femora and tibiae of transgenic mice by flushing with PBS. The megakaryocytes were enriched by culturing BMCs in IMDM with 10% FBS, streptomycin/penicillin and 50 ng/ml TPO for 5 days. For some experiments, the erythrocytes were removed from the isolated BMCs by incubation, for 2 min on ice, with 2 ml of Red Blood Cell (RBC) lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA pH 7.4).
Megakaryocytic differentiation and maturation in cell lines and BMCs
For MKs differentiation and maturation, cell line cultures were treated with 20 nM phorbol 12-myristate 13-acetate (PMA, Sigma) for 10 days. BMCs were incubated with 50 ng/ml recombinant mouse thrombopoietin (TPO, Miltenyi) in combination with 10 ng/ml Stem Cell Factor (SCF, Miltenyi), 10 ng/ml Interleukin-3 (IL-3, Invitrogen), 10 ng/ml IL-11 (Miltenyi) and 10 ng/ml IL-6 (Miltenyi) for 6 days.
K562 cells were permanently transfected with pLTR2-C3G , pSuper-C3Gi  and CRISPR-C3G (Santa Cruz Biotechnology, Inc.) and the corresponding empty vectors (pLTR2-CT, pSuper-CT and CRISPR-CT), for up-regulation, down-regulation or abrogation of C3G expression, respectively, using the Gene Pulser Electroporation System (Bio-Rad). Additionally, the pLTR2 clones were co-transfected with pSuper.gfp/neo vector to express GFP fluorescence (pLTR2-CT/GFP and pLTR2-C3G/GFP clones). These clones were maintained with complete medium supplemented with 1:20 Killer Hat solution , 250 μg/ml neomycin or 2 μg/ml puromycin, as appropriate.
The expression of C3G in HEL cells was downregulated by infection with second generation lentiviral particles, which were generated by transient transfection of HEK-293 T cells with psPAX2, pMD2.G and pLVTHM (-CT or -C3Gi) vectors (Addgene), using PEI transfection protocol. HEL cells were infected with the lentiviral particles containing the shRNA of C3G at a MOI of 25. Finally, GFP+ cells were selected by single-cell isolation and clonal expansion. The shRNA used to downregulate C3G expression was: 5′- CCACTATGATCCCGACTAT-3′.
Permanent GATA-1 silencing in Dami cells was performed by infection with human GATA-1 shRNA lentiviral particles (75,000 infectious units) containing a mixture of different shRNAs (Santa Cruz Biotechnology sc-35,452-V) in the presence of 10 μg/ml Polybrene (Santa Cruz Biotechnology sc-134,220). Cells were selected with puromycin (1 μg/ml).
Cell lysis and Western blot
Cell line cultures were lysed using Cell Lysis buffer (Cell Signaling Technology) supplemented with 25 μM NaF and 1 mM PMSF. Total protein lysates were boiled with 2x Laemmli buffer before being resolved on SDS-PAGE. PVDF membranes were blotted using the following antibodies: C3G H-300 (sc-15,359), p-C3G Tyr504 (sc-12,926), α-globin H-80 (sc-21,005), ERK K-23 (sc-94) and GATA-1 N6 (sc-265) from Santa Cruz Biotechnologies, β-tubulin (T5293) and β-actin (A5441) from Sigma and p21 Waf1/Cip1 (2947) from Cell Signaling Technology.
RNA was reverse transcribed using SuperScript™ III First-Strand Synthesis System (Thermofisher) to generate DNA. Semi-quantitative PCR was performed using BioTaq™ polymerase (Bioline) using the following primers: for GPA: forward 5´-GGAATTCCAGCTCATGATCTCAGGATG-3′ and reverse 5´-TCCACATTTGGTTTGGTGAACAGATTC-3′; for CD61: forward 5´-TATAGCATTGGACGGAAGGC-3′ and reverse 5´-GACCTCATTGTTGAGGCAGG-3′; for GAPDH: forward 5´-TGCACCACCAACTGCTTAGC-3′ and reverse 5′- TCTTCTGGGTGGCAGTGATG-3′.
Analysis of cell surface markers and DNA content by flow cytometry
Megakaryocytic markers, CD41 and CD61, and erythroid marker (GPA) were analyzed by flow cytometry using specific fluorochrome-conjugated antibodies: anti-human antibodies for human cell lines from Immunostep (CD41-PE, CD61-APC and GPA-FITC or GPA-PE) and anti-mouse antibodies for mouse BMCs from eBiosciences (CD41-APC and CD61-PE). After differentiation treatment, washed cells were incubated in 100 μl of PBS with 0.5–1 μg of indicated antibodies for 20 min on ice. Then, cells were washed once and the fluorescence was measured using FACSCalibur™ and BD Accuri™ cytometers and analyzed using FlowJo and Accuri software, respectively.
To determine the ploidy status, after 6–10 days of differentiation, cultures of cell lines and BMCs were harvested, washed with PBS and fixed with ethanol by the addition of 0.5 ml of 70% ethanol dropwise. After incubation for at least 1 h, cells were washed twice with PBS and stained with anti-CD41-FITC antibody from BD Pharmigen (BMCs only), as described before, and subsequently incubated with a mixture of 100 μg/ml RNase (Promega) and 50 μg/ml Propidium Iodide (PI, Sigma) in PBS for at least 40 min at RT in the dark. For the analysis, CD41+ cells were gated and cell doublets were excluded from the analysis. The ploidy distribution of the CD41+ populations was determined using a BD Accuri™ cytometer.
Confocal immunofluorescence microscopy and may-Grunwald-Giemsa staining
3 × 106 cells were seeded onto 6 cm plates containing glass coverslips pre-coated with 5 μg/ml of fibronectin (Sigma). Cells were incubated with serum-free media for 48 h, which allowed for enhanced cell attachment, and then fixed with 3.7% paraformaldehyde (PFA, Sigma) for 15 min. Fixed cells were washed twice with PBS, permeabilized with 0.1% Triton X-100 (Sigma), and washed again with PBS. Coverslips were blocked with 2% BSA for 1 h, and then incubated for 1 h at RT with primary antibody C3G #1008 , followed by incubation with Cy5-anti-rabbit antibody. Nuclei were stained with DAPI for 10 min. Coverslips were mounted with Mowiol® (Calbiochem). Images were obtained at the same exposure time with a Leica TCS SP5 confocal microscope and pictures were processed using LSM Image Browser, ImageJ Software and ZEN lite Imaging Software.
For Giemsa staining, after 48 h of PMA treatment, cells were centrifuged onto glass coverslips for 3 min at 500 g to allow cell attachment. Then, cells were fixed with 100% ethanol and the slides were stained with May-Grunwald-Giemsa.
Rap1 activity assay by immunofluorescence
Preparation of samples for detection of active Rap1 by confocal microscopy was performed essentially as described above, with some modifications [27, 28]. Aliquots of 1.5 × 106 cells in PBS were treated with 20 nM PMA at indicated times and then immediately fixed by adding 4% PFA for 20 min at RT. Cells were washed with PBS by centrifugation and permeabilized with 0.2% Triton + 1% BSA for 5 min at RT. Cells were then incubated with 0.3 mg/ml GST-RalGDS-RBD purified protein for 45 min at RT, washed three times with PBS and incubated with anti-GST for 1 h at RT. Cells were washed three times with PBS, and incubated with Cy3 or Cy5-conjugated anti-mouse antibodies for 1 h at RT. Nuclei were stained with DAPI for 10 min. After washing, cells were centrifuged onto glass coverslips for 3 min at 500 g and mounted with Mowiol. Negative controls were performed as follows: (1) without the GST-RalGDS-RBD protein, as control of the specificity of the anti-GST primary antibody; (2) by replacing GST-RalGDS-RBD with GST alone at the same molarity; (3) without anti-GST primary antibody, to detect any non-specific staining by the secondary antibody. For active Rap1, z-sections of 0.25 μm were acquired using Leica TCS SP5 confocal microscope. All images were obtained at the same exposure time and processed using LSM Image Browser and ImageJ software.
A commercial collagen-based system (MegaCult-C, StemCell Technologies Inc.) was used to assay colony-forming units (CFUs) of mouse megakaryocyte progenitors. Briefly, 2.2 × 106 freshly isolated BM cells were resuspended into 1 ml IMDM medium (33x of the final cell concentration). Then, 50 μl of this cell suspension was mixed with 150 μl of IMDM, containing cytokines at 11x of the final concentration (1x: 50 ng/ml TPO, 20 ng/ml IL-6, 50 ng/ml IL-11 and 10 ng/ml IL-3) and 850 μl of MegaCult™ medium. Finally, 600 μl of cold collagen was added (≈1600 μl) and 750 μl of this final cell suspension was cultured into the two wells of a double chamber slide (μ-Slide 2 well, Ibidi), each containing ≈50,000 cells. Cultures were maintained at 37 °C and 5% CO2 for 8 days. Collagen gels were dehydrated, fixed and stained according to the manufacturer’s specifications. Acetylchorinesterase-positive colonies with 3 or more MKs were scored as CFU-MKs.
Time lapse analysis of bone marrow explants
Intact marrow was obtained by flushing mouse femora with Tyrode’s-HEPES buffer (134 mM NaCl, 0.34 mM NaHPO4, 2.9 mM KCl, 12 mM NaHCO3, 20 mM HEPES), 5 mM Glucose, 0.35% Albumin, 1 mM MgCl2, 2 mM CaCl2 and 10 U/ml Penicillin/Streptomycin) using a 21-gauge needle. The marrows were cut into 0.5–1 mm thick transverse sections with a surgical blade, under a binocular microscope. The explants were placed in an incubation chamber (μ-Slide 8 well IbiTreat, Ibidi) with Tyrode’s-HEPES buffer containing 5% mouse serum and were maintained at 37 °C for 6 h. MKs at the periphery of the explant were monitored under an inverted microscope (Nikon Eclipse TE2000-E), coupled to a video camera (Hamamatsu Orca-er). The images were sequentially acquired at 10 min intervals for 6 h and then mounted and processed using ImageJ and Metamorph software.
To identify the MKs in the periphery of the explant, anti-mouse CD41-APC antibody (eBiosciences) was added to the Tyrode’s-HEPES buffer prior to placing the explants in the incubation chamber. MKs were classified according to the morphology: i) spherical megakaryocytes, ii) megakaryocytes with protrusion and iii) megakaryocytes with proplatelets .
Isolation of cells from the bone matrix
To analyze the MKs associated to the osteoblastic niche, after isolation of BMCs from the femur, the remaining bones were cut into 1 mm pieces and incubated with 1 mg/ml collagenase Type I (Sigma) and 1 mg/ml dispase Type II (Sigma) at 37 °C for 2 h under vigorous stirring to detach the cells most tightly adhered to the bone matrix.
Analysis of the number of platelets by flow cytometry
Blood (100–200 μl) was collected by submandibular puncture from anesthetized mice (isoflurane), and anticoagulated with EDTA. Blood was washed with same volume of Tyrode’s-HEPES buffer plus 5 mM glucose, and stained with anti-CD41-APC antibody for 15 min at RT. The number of platelets was determined by measuring 50 μl of blood using a BD Accuri ™ cytometer.
Platelet production in vivo in response to TPO
Mouse TPO (Molecular Innovations®) was administered by intraperitoneal injection (5 μg per mouse). Platelet number was determined, at the indicated time points, as described above. Bone marrow cells were harvested 11 days after injection and the percentage of megakaryocytes was analyzed by flow cytometry.
Platelet clearance analysis
Mice were injected, through the lateral tail vein, with 600 μg of hydroxysuccinimido-biotin (NHS-biotin, Sigma) in 200 μl of buffer containing 140 mM NaCl and 10% DMSO. At the indicated time points, 20 μl of whole blood, from submandibular puncture, was mixed with 200 μl ml BSGC buffer (116 mM NaCl, 13.6 mM tri-sodium citrate, 8.6 mM Na2HPO4, 1.6 mM KH2PO4, 0.9 mM EDTA, 11,1 mM glucose) and 1 ml of balanced salt solution (BSS; 149 mM NaCl, 3.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 7.4 mM HEPES, 1.2 mM KH2PO4, 0.8 mM K2HPO4, 3% FBS). Cells were pelleted at 1400 g for 5 min and resuspended in PBS. Platelets were stained with CD41-APC for 15 min, followed by PE-Streptavidin for 1 h on ice. Biotinylated platelets were washed in BSS buffer and analyzed by flow cytometry.
Tumor implantation and analysis of platelets, MKs and adipocytes
1 × 106 B16-F10 melanoma cells in PBS were subcutaneously injected into Tg-C3G and WT-C3G mice. After 15 days of tumor growth, the BM was extracted, fixed in 4% formaldehyde, embedded in paraffin and stained with H&E. The number of MKs was quantified using an Ariol IHC Scanner, based on the Area_score and the Intensity_score parameters (Leyca Biosystems). The number of adipocytes was determined using the Adiposoft software (ImageJ), which discriminates particles between 4 and 30 μm. A visual double-check was done to eliminate the false-positive results.
Data have been represented as the mean ± SEM (Standard Error of the Mean) or the median ± SEM values of at least 3 independent experiments from each genotype. The Kolmogorov-Smirnov test was performed to determine if data fit into a normal distribution. To compare between two experimental groups, unpaired Student’s t-test was computed, when the data were normally distributed. The Mann Whitney’s U-test was computed as a non-parametric procedure when our data were not normally distributed.
In fluorescence measurements in cell lines cultures, due to high variability in fluorescence intensities between independent experiments, the data were normalized against the control values. To calculate the significance between the different experimental conditions, a two-way ANOVA test was performed. Then, Holm-Sidak post-hoc pairwise analysis was calculated to determine the significant differences in groups two to two.