TPO induces the BMP4 autocrine loop in MK progenitors. Purified CD34⁺ cells (6 × 10⁵/mL) were cultured in 4GF serum-free medium supplemented with TPO or BMP4 (100 ng/mL). (A) Cells were incubated in the presence of type Ia (sBMPR-Ia, 1 μg/mL) and type Ib (sBMPR-Ib, 4 μg/mL) soluble BMP receptors to inhibit the BMP signaling pathway as described.¹³  After 3 days of culture, wells were assayed for their CFU-MK and after 7 days for their platelet content. Results represent the mean value plus or minus SEM of, respectively, 6 and 4 experiments. (B) RT-PCRq expression data are expressed as a mean ratio of the gene of interest to 2 control genes, TBP and COF. Results are expressed as a fold increase calculated from the ratio of TPO-treated cells to nontreated cells. Therefore, the 1 value represented on the graph matches the baseline level of the gene in nontreated cells. Results represent the mean value plus or minus SEM of 5 independent experiments. (C) After 7 days of culture, the supernatant was harvested and cleared by 10 minutes of centrifugation at 3000g, then placed in 96-well plates for ELISA quantification of BMP4 as described in “BMP4 and PF4 quantification.” Results expressed in pg/mL of BMP4 represent the mean value plus or minus SEM of 4 to 9 experiments. * indicates statistically significant difference from those in nontreated cells (P < .05).