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Blood, Vol. 112, Issue 8, 3154-3163, October 15, 2008
BMP4 regulation of human megakaryocytic differentiation is involved in thrombopoietin signaling
Blood Jeanpierre et al.
112: 3154
Supplemental materials for: Jeanpierre et al
Files in this Data Supplement:
- Table S1. Oligonucleotide primers used for human gene expression analysis by RT-PCR (PDF, 11.1 KB)
- Table S2. Oligonucleotide primers used for human gene expression analysis by QT-PCR (PDF, 10.3 KB)
- Table S3. Human FOG2 and Fli1 expressions analysis by QT-PCR in hematopoietic cell populations (PDF, 10.8 KB)
- Figure S1. Cell surface markers, gene, and protein expression during in vitro megakaryopoieis (JPG, 201 KB)
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CD34+ cells were cultured as described in the Methods. At the indicated times throughout the experiment, cells were harvested and analyzed by flow cytometry, RT-PCR and western blot. (A) Phenotypic analysis of early MK-specific markers CD41, CD61, the erythro-MK common marker CD36 and the erythroid-specific Glycophorin A (GPA), and (B) of late MK markers CD42a, CD42b and TPO-R (c-mpl) was performed. Results are presented as percentages of positive viable cells ± SEM of an average of 10 independent experiments. (C) PF4 and CD42a expression in MK cells during differentiation were analyzed by western blot. Total cell lysates obtained from cells at various times of culture were quantified and 10 µg of total proteins were separated by SDS-polyacrylamide gel electrophoresis. Immunodetection was performed as described in the Methods using monoclonal mouse anti-PF4 and anti CD42a, with anti-Actin as a loading control. (D) RT-PCR was used to analyze the expression of various genes at different time points. In all conditions, we found a similar level of expression of the housekeeping GAPDH gene, which allowed us to compare levels of gene expression between the different cell types. Act R, Activin receptor; BMP R, BMP receptor; EpoR, Erythropoietin Receptor ; FOG, Friend of GATA; Fli-1, Friend Leukemia Integration 1; GAPDH, Glyceraldehyde-3-Phosphate Dehydrogenase; PF4, Platelet Activation Factor 4; TPO-R, Thrombopoietin Receptor (c-mpl).
- Figure S2. Receptors, cell surface markers, and cell adhesion during in vitro megakaryopoieis (JPG, 37 KB)
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Bone marrow cells were selected for CD34+ cells by immunomagnetic selection (A) The expression of the different BMPR at day 0 on these CD34+ purified cells has been quantified by RT-PCRq. Data for BMPR expression are presented as a mean ratio of the gene of interest to control COF gene. Results are expressed as a fold increase of the ratio of sorted cells to human mammary fibroblasts used as non hematopoietic reference cells. Results represent the mean value ± SEM of 3 independent experiments. Purifed CD34+ cells (6 × 105/ml) were incubated in 4GF serum-free medium for 3 to 18 days in the presence or not (None) of 50ng/ml TPO or BMP4. (B) At the indicated times throughout the experiment, cells were harvested and analyzed by flow cytometry with gating on viable cells for the expression of the early MK-specific marker CD41 and the late MK marker CD42b. Results are presented as percentages of positive viable cells ± SEM of 8 experiments for addition of TPO or BMP4. (C) Cells (5 × 104/well) were allowed to adhere to fibronectin (50 µg/ml) for 1 hour in the presence of the indicated protein (50ng/ml) at 37°C in adhesion buffer (PBS with 0.03% BSA), and the number of CFC in both non-adherent and adherent fractions was determined as described.10 Results, expressed as the % of adherent CFC calculated as follows: (adherent CFC number/adherent+non-adherent CFC number) ×100; represent the mean value ± SEM of 6 experiments on different bone marrow samples.
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