American Society of Hematology

Figure 2.

$Figure 2. Erythroid colony formation and proliferation of K-Ras-/- and WT c-kit+ day-13.5 fetal liver cells in response to EPO and KitL. (A) Frequency of erythroid progenitors in WT and K-Ras-/- nonfractionated day-13.5 fetal liver cells. Total nucleated fetal liver cells (40 000 cells/mL) were plated for growth of BFU-Es in methylcellulose medium supplemented with 10 ng/mL KitL and 5 U/mL EPO. For CFU-Es, total nucleated fetal liver cells (40 000 cells/mL) were plated in methylcellulose medium supplemented with 5 U/mL EPO. CFU-Es and BFU-Es were counted by indirect microscopy at days 2 and 7 of culture, respectively. Results represent the mean number of colonies and error bars represent the standard error of the mean of 5 parallel independent experiments, which were performed in triplicate from embryos isolated from the same litter. *P < .05 by Student paired t test. (B) Frequency of erythroid progenitors in c-kit+–sorted cells isolated from day-13.5 WT and K-Ras-/- fetal livers. C-kit+ cells were isolated by immunomagnetic bead enrichment and sorted as described in “C-kit+ cell isolation” to a purity of more than 85% as tested by fluorescence cytometry (data not shown). C-kit+ cells (4000 cells/mL) were plated for growth of BFU-Es in methylcellulose medium containing various concentrations of KitL as indicated combined with a single concentration of EPO (5 U/mL). C-kit+ cells (4000 cells/mL) were plated for growth of CFU-Es in methylcellulose medium containing various concentrations of EPO alone as indicated. CFU-Es and BFU-Es were counted by indirect microscopy at days 2 and 7 of culture, respectively. Results represent the mean number of colonies per 4000 c-kit+ ± SEM of 5 parallel independent experiments. Colony assays were performed in triplicate from c-kit+ cells isolated from embryos isolated from the same litter. *P < .05 by Student paired t test. (C) Proliferation of WT and K-Ras-/- c-kit+ day-13.5 fetal liver cells in response to no growth factors, EPO and KitL alone, or in combination. Freshly isolated c-kit+ fetal liver cells were plated in a 96-well plate at a concentration of 10 000 cells/well in serum-free medium with no additional growth factors or 10 ng/mL KitL and 5 U/mL EPO in combination or alone. After 48 hours of culture, cells were pulsed with tritiated thymidine and harvested 16 hours later for measurement of β emission. CPM indicates counts per minute. Results represent the mean thymidine incorporation ± SEM of 5 independent experiments. Thymidine incorporation assays were performed in triplicate from c-kit+ cells isolated from embryos harvested from the same litter. *P < .05 by Student paired t test.$

Erythroid colony formation and proliferation of K-Ras^-/- and WT c-kit⁺ day-13.5 fetal liver cells in response to EPO and KitL. (A) Frequency of erythroid progenitors in WT and K-Ras^-/- nonfractionated day-13.5 fetal liver cells. Total nucleated fetal liver cells (40 000 cells/mL) were plated for growth of BFU-Es in methylcellulose medium supplemented with 10 ng/mL KitL and 5 U/mL EPO. For CFU-Es, total nucleated fetal liver cells (40 000 cells/mL) were plated in methylcellulose medium supplemented with 5 U/mL EPO. CFU-Es and BFU-Es were counted by indirect microscopy at days 2 and 7 of culture, respectively. Results represent the mean number of colonies and error bars represent the standard error of the mean of 5 parallel independent experiments, which were performed in triplicate from embryos isolated from the same litter. ^*P < .05 by Student paired t test. (B) Frequency of erythroid progenitors in c-kit⁺–sorted cells isolated from day-13.5 WT and K-Ras^-/- fetal livers. C-kit⁺ cells were isolated by immunomagnetic bead enrichment and sorted as described in “C-kit⁺ cell isolation” to a purity of more than 85% as tested by fluorescence cytometry (data not shown). C-kit⁺ cells (4000 cells/mL) were plated for growth of BFU-Es in methylcellulose medium containing various concentrations of KitL as indicated combined with a single concentration of EPO (5 U/mL). C-kit⁺ cells (4000 cells/mL) were plated for growth of CFU-Es in methylcellulose medium containing various concentrations of EPO alone as indicated. CFU-Es and BFU-Es were counted by indirect microscopy at days 2 and 7 of culture, respectively. Results represent the mean number of colonies per 4000 c-kit⁺ ± SEM of 5 parallel independent experiments. Colony assays were performed in triplicate from c-kit⁺ cells isolated from embryos isolated from the same litter. ^*P < .05 by Student paired t test. (C) Proliferation of WT and K-Ras^-/- c-kit⁺ day-13.5 fetal liver cells in response to no growth factors, EPO and KitL alone, or in combination. Freshly isolated c-kit⁺ fetal liver cells were plated in a 96-well plate at a concentration of 10 000 cells/well in serum-free medium with no additional growth factors or 10 ng/mL KitL and 5 U/mL EPO in combination or alone. After 48 hours of culture, cells were pulsed with tritiated thymidine and harvested 16 hours later for measurement of β emission. CPM indicates counts per minute. Results represent the mean thymidine incorporation ± SEM of 5 independent experiments. Thymidine incorporation assays were performed in triplicate from c-kit⁺ cells isolated from embryos harvested from the same litter. ^*P < .05 by Student paired t test.

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