Figure 2.
Figure 2. Myeloid progenitor development by B-raf–/– ES cells cocultured with OP9 stromal cells in vitro. (A) The percentage of mesodermal colonies developed each day after plating equal numbers of undifferentiated ES cells on OP9 cells is indicated for days 1 to 5 of coculture. Inset figure shows the morphology of a typical mesodermal colony observed using a phase-contrast microscope (original magnification, × 100). The data represent the average ± SD for 3 independent experiments. (B) The number of day-8 hematopoietic clusters generated per 1 × 105 day-5 cells plated is presented for wild-type (+/+) and B-raf–/– (–/–) ES cells (P = .033). Inset figure shows a typical hematopoietic cluster (original magnification, × 200). Data are the average ± SD for 4 independent experiments. Images in panels A and B were obtained using a Nikon Eclipse TE300 microscope (Nikon, Melville, NY), IPLab Spectrum imaging software (Scanalytics, Fairfax, VA), and Photoshop (Adobe Systems, San Jose, CA). (C) The bar graph shows the number of myeloid progenitors obtained after plating 1 × 104 day-9 loosely adherent and floating cells generated from wild-type (+/+) and B-raf–/– (–/–) ES cells (P = .015). Data are the average ± SD for 3 independent experiments. (D) Western blot analysis of B-Raf expression. Cell lysates were prepared from undifferentiated ES cells (day 0) and OP9 cocultured differentiating ES cells (day 5 and day 9), fractionated by SDS-PAGE and immunoblotted with a B-Raf–specific antibody. Cell lysates from undifferentiated (day 0) B-raf–/– ES cells, mouse embryo fibroblasts (MEF), and OP9 cells serve as controls. The membrane was reblotted with β-tubulin–specific antibody to confirm equal loading. (E) Western blot analysis of serum-stimulated (20%; 0 to 60 minutes) undifferentiated day-0 ES cells. Cell lysates were fractionated by SDS-PAGE and immunoblotted for phosphorylated ERK1/2 and total ERK1/2. (F, top) Western blot analysis of serum-stimulated (20%; 0-60 minutes) differentiating ES cells on day 5. Cell lysates were fractionated by SDS-PAGE and immunoblotted for phosphorylated ERK1/2 and total ERK1/2. (Middle) Western blot for phospho- and total p38 performed as in the top panel. (Bottom) Quantification of ERK phosphorylation in serum-stimulated day-5 mesodermal colony cells is presented. The immunoblots were visualized using chemiluminescence and x-ray film, and the bands were quantified using NIH image software. Relative intensity of serum-induced ERK phosphorylation is expressed as a percentage of the peak (10-minute stimulation) level of wild-type cells (100%). The data are the average ± SD of 4 independent experiments. Basal ERK phosphorylation level in B-raf–/– cells was significantly decreased compared with wild-type cells (*P = .042).

Myeloid progenitor development by B-raf–/– ES cells cocultured with OP9 stromal cells in vitro. (A) The percentage of mesodermal colonies developed each day after plating equal numbers of undifferentiated ES cells on OP9 cells is indicated for days 1 to 5 of coculture. Inset figure shows the morphology of a typical mesodermal colony observed using a phase-contrast microscope (original magnification, × 100). The data represent the average ± SD for 3 independent experiments. (B) The number of day-8 hematopoietic clusters generated per 1 × 105 day-5 cells plated is presented for wild-type (+/+) and B-raf–/– (–/–) ES cells (P = .033). Inset figure shows a typical hematopoietic cluster (original magnification, × 200). Data are the average ± SD for 4 independent experiments. Images in panels A and B were obtained using a Nikon Eclipse TE300 microscope (Nikon, Melville, NY), IPLab Spectrum imaging software (Scanalytics, Fairfax, VA), and Photoshop (Adobe Systems, San Jose, CA). (C) The bar graph shows the number of myeloid progenitors obtained after plating 1 × 104 day-9 loosely adherent and floating cells generated from wild-type (+/+) and B-raf–/– (–/–) ES cells (P = .015). Data are the average ± SD for 3 independent experiments. (D) Western blot analysis of B-Raf expression. Cell lysates were prepared from undifferentiated ES cells (day 0) and OP9 cocultured differentiating ES cells (day 5 and day 9), fractionated by SDS-PAGE and immunoblotted with a B-Raf–specific antibody. Cell lysates from undifferentiated (day 0) B-raf–/– ES cells, mouse embryo fibroblasts (MEF), and OP9 cells serve as controls. The membrane was reblotted with β-tubulin–specific antibody to confirm equal loading. (E) Western blot analysis of serum-stimulated (20%; 0 to 60 minutes) undifferentiated day-0 ES cells. Cell lysates were fractionated by SDS-PAGE and immunoblotted for phosphorylated ERK1/2 and total ERK1/2. (F, top) Western blot analysis of serum-stimulated (20%; 0-60 minutes) differentiating ES cells on day 5. Cell lysates were fractionated by SDS-PAGE and immunoblotted for phosphorylated ERK1/2 and total ERK1/2. (Middle) Western blot for phospho- and total p38 performed as in the top panel. (Bottom) Quantification of ERK phosphorylation in serum-stimulated day-5 mesodermal colony cells is presented. The immunoblots were visualized using chemiluminescence and x-ray film, and the bands were quantified using NIH image software. Relative intensity of serum-induced ERK phosphorylation is expressed as a percentage of the peak (10-minute stimulation) level of wild-type cells (100%). The data are the average ± SD of 4 independent experiments. Basal ERK phosphorylation level in B-raf–/– cells was significantly decreased compared with wild-type cells (*P = .042).

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