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Blood, Vol. 114, Issue 1, 153-156, July 2, 2009
Survivin is not required for the endomitotic cell cycle of megakaryocytes
Blood Wen et al.
114: 153
Supplemental materials for: Wen et al
Genotyping
For genotyping the PF4-Cre transgene, the PF4-Cre forward (5-gcctgcattaccggtcgatgcaacga-3) and PF4-Cre reverse (5-gtggcagatggcgcggcaacaccatt-3) primers were used to amplify a 725-bp PCR product. Three primers (Adv17, Adv25, and Adv28) were used to genotype the wild-type, floxed, and Cre deleted survivin allele as previously described (1). Anti-CD42b antibody induction of transient thrombocytopenia
PF4-Cre/Surfl∕fl and Surfl∕fl mice were administered anti-CD42b antibody (Emfret Analytics, Germany) at 2µg/g body weight in 200 µl by intraperitoneal injection. Peripheral blood was collected from the tail vein at 24-hour intervals from the day of antibody treatment (pre-treatment) until 144 h after treatment. Platelet counts and other peripheral blood values were determined with a Hemavet HV950FS multi-species hematology instrument (Drew Scientific, Inc., Oxford, CT). Flow cytometry
Megakaryocytes were stained with anti-CD41 (BD Pharmingen, San Diego, CA) and anti-CD42 (Emfret Analytics, Germany) antibodies conjugated to FITC and PE respectively. For DNA content and apoptosis assays, cells were stained with 4,6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, MO) and APC conjugated annexin-V (BioVision, Mountain View, CA) antibodies. Surface marker expression, DNA content and annexin-V staining were acquired using a LSR II flow cytometer (BD Biosciences, San Jose, CA) and data were analyzed with FlowJo software (Tree Star, Ashland, OR) Immunofluorescence
Lineage-depleted Surfl∕fl bone marrow progenitors were infected by spinoculation on days 2 and 3 of expansion with retroviruses harboring MSCV-GFP or MSCV–Cre-GFP prior to differentiation with TPO at 20 ng/ml. After 2 days of differentiation, cells were cytospun onto slides and fixed in 4% fresh paraformaldehyde at room temperature for 20 minutes. Cells were then washed and permeabilized with 0.3% Triton X-100 in PBS for 20 minutes at room temperature. 10% bovine serum albumin in PBS was used to block nonspecific binding for 60 minutes at room temperature followed by 1-hour incubation with primary antibody (α-tubulin, Sigma T6199, 1:1000 dilution) at room temperature. Slides were incubated with Alexa Fluor 594–conjugated secondary antibody, washed and stained with DAPI to visualize chromosomes. Images were acquired with Nikon C1Si spectral laser scanning confocal microscope and an Olympus Disk Scanning Unit, inverted IX81 automated microscope. REFERENCE 1. Leung CG, Xu Y, Mularski B, Liu H, Gurbuxani S, Crispino JD. Requirements for survivin in terminal differentiation of erythroid cells and maintenance of hematopoietic stem and progenitor cells. J Exp Med. 2007;204:1603–11.
Files in this Data Supplement:
- Table S1. Comparison of hematological parameters of peripheral blood collected from PF4Cre/Sur fl/fl versus Surfl∕fl or Surfl∕+ mice (PDF, 22.7 KB)
- Figure S1. Recovery of platelet counts after treatment with CD42b antibody (JPG, 97.3 KB)
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Surfl∕fl and PF4-Cre/Surfl∕fl mice received 2 µg/g body weight anti-CD42b antibody to induce transient thrombocytopenia. Platelet recovery was measured by performing complete blood counts at the indicated time points. Results are shown as mean ± SEM, with 7 mice for each group.
- Figure S2. Ex vivo deletion of survivin from bone marrow cells has no effect on polyploidization or differentiation of bone marrow megakaryocytes in liquid culture with TPO (JPG, 392 KB)
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Bone marrow cells from Surfl∕fl (or Surfl∕+) and PF4-Cre/Surfl∕fl mice were cultured ex vivo in the presence of TPO (20 ng/ml) for 3 days. Cells were then analyzed by flow cytometry for (A) CD41 expression, (B) CD42 expression, (C) DNA content, and (D) Annexin V staining. Note that DNA content and Annexin V staining are shown for only the CD41+ population. In (E), DNA content is shown for BSA gradient purified megakaryocytes generated from bone marrow cell culture in the presence of TPO for 3 days. Data are derived from 5–6 mice for each group. (F) Deletion of the floxed region was monitored by multiplex PCR using three primers to amplify the floxed (fl) and excised (ex) alleles. PCR of tail, bone marrow cells (BM) before and after liquid culture with TPO, and BSA gradient purified megakaryocytes (BSA purified MKs) from two representative experiments are shown.
- Figure S3. Survivin deletion affects mitosis of low ploidy cells, but not of high ploidy cells (JPG, 313 KB)
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(A) α-tubulin and DAPI stained confocal images of low (a) and high (b) ploidy Surfl∕fl cells transduced with retroviruses expressing either Cre-GFP or GFP alone. No differences were observed in terms of chromosome separation between the Cre-GFP and GFP transduced high ploidy cells. In contrast, the majority of the Cre-GFP transduced cells exhibited a failure of chromosome separation, consistent with the known requirement for survivin in cytokinesis. All images were taken with 100× oil immersion objective.
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