1. Activation of phosphatidylinositol 3-kinase is important for erythropoietin-induced erythropoiesis from CD34+ hematopoietic progenitor cells 
June Helen Myklebust, Heidi Kiil Blomhoff, Leiv Sindre Rusten, Trond Stokke, Erlend B Smeland 
Experimental Hematology 
Volume 30, Issue 9, Pages (September 2002)
DOI: /S X(02)

2. Figure 1
LY (LY) and staurosporine (ST) inhibit the EPO-induced formation of erythroid cells (GPA+ cells) from CD34+ hematopoietic progenitor cells. CD34+ (100,000–150,000 cells/well) were cultured in X-VIVO 15 complete medium in the presence of EPO (5 U/mL) with or without LY (50 μmol/L) or ST (20 nmol/L). After 6 days of culture, the cells were immunophenotyped. One representative experiment of four is shown.
Experimental Hematology  , DOI: ( /S X(02) )

3. Figure 2
Effect of LY (LY) or staurosporine (ST) on the phenotype of CD34+ cells stimulated with EPO. CD34+ cells (100,000–150,000 cells/well) were cultured in X-VIVO 15 complete medium in the presence of EPO with or without LY (50 μmol/L) or ST (20 nmol/L). After 6 days of culture, the cells were analyzed by flow cytometry. GPA = glycophorin A. Mean ± SEM of 4 to 8 experiments. (A) Percentage of GPA+ cells. * p < 0.001; n = 8, # p < 0.02; n = 4. (B) Percentage of CD34+ cells. *p < 0.004, n = 8. #p < 0.021, n = 4. (C) Percentage of CD13+ cells. *p < 0.018, n = 4.
Experimental Hematology  , DOI: ( /S X(02) )

4. Figure 2
Effect of LY (LY) or staurosporine (ST) on the phenotype of CD34+ cells stimulated with EPO. CD34+ cells (100,000–150,000 cells/well) were cultured in X-VIVO 15 complete medium in the presence of EPO with or without LY (50 μmol/L) or ST (20 nmol/L). After 6 days of culture, the cells were analyzed by flow cytometry. GPA = glycophorin A. Mean ± SEM of 4 to 8 experiments. (A) Percentage of GPA+ cells. * p < 0.001; n = 8, # p < 0.02; n = 4. (B) Percentage of CD34+ cells. *p < 0.004, n = 8. #p < 0.021, n = 4. (C) Percentage of CD13+ cells. *p < 0.018, n = 4.
Experimental Hematology  , DOI: ( /S X(02) )

5. Figure 2
Effect of LY (LY) or staurosporine (ST) on the phenotype of CD34+ cells stimulated with EPO. CD34+ cells (100,000–150,000 cells/well) were cultured in X-VIVO 15 complete medium in the presence of EPO with or without LY (50 μmol/L) or ST (20 nmol/L). After 6 days of culture, the cells were analyzed by flow cytometry. GPA = glycophorin A. Mean ± SEM of 4 to 8 experiments. (A) Percentage of GPA+ cells. * p < 0.001; n = 8, # p < 0.02; n = 4. (B) Percentage of CD34+ cells. *p < 0.004, n = 8. #p < 0.021, n = 4. (C) Percentage of CD13+ cells. *p < 0.018, n = 4.
Experimental Hematology  , DOI: ( /S X(02) )

6. Figure 3
Activation of Akt is inhibited by LY (LY) in CD34+ cells. CD34+ cells were cultured overnight in X-VIVO 15 complete medium, followed by incubation with LY (50 μmol/L) for 30 minutes before stimulation with EPO (10 U/mL) for another 20 minutes. Cells were lysed and 75% of the cell lysate was immunoprecipitated with anti-Akt antibody. The immunoprecipitates were subjected to an in vitro kinase assay using GSK-3 as a substrate (1 μg). Reaction products were resolved by 12% SDS-PAGE and subjected to immunoblot analysis using anti-phospho–GSK-3 α/β antibody. The remaining 25% of the cell lysate was run on a 12% SDS-PAGE and hybridized with actin Ab. (A) One representative experiment of three is shown. (B) Relative expression of phospho–GSK-3 α/β, determined by densitometric scanning of hyperfilms. Mean ± SEM of three experiments.
Experimental Hematology  , DOI: ( /S X(02) )

7. Figure 3
Activation of Akt is inhibited by LY (LY) in CD34+ cells. CD34+ cells were cultured overnight in X-VIVO 15 complete medium, followed by incubation with LY (50 μmol/L) for 30 minutes before stimulation with EPO (10 U/mL) for another 20 minutes. Cells were lysed and 75% of the cell lysate was immunoprecipitated with anti-Akt antibody. The immunoprecipitates were subjected to an in vitro kinase assay using GSK-3 as a substrate (1 μg). Reaction products were resolved by 12% SDS-PAGE and subjected to immunoblot analysis using anti-phospho–GSK-3 α/β antibody. The remaining 25% of the cell lysate was run on a 12% SDS-PAGE and hybridized with actin Ab. (A) One representative experiment of three is shown. (B) Relative expression of phospho–GSK-3 α/β, determined by densitometric scanning of hyperfilms. Mean ± SEM of three experiments.
Experimental Hematology  , DOI: ( /S X(02) )

8. Figure 4
Time course of LY (LY)-mediated inhibition of EPO-induced or EPO- and SCF-induced formation of erythroid cells from CD34+ progenitor cells or from CD34+CD71+CD45RA− erythroid progenitors. CD34+ cells were immunophenotyped immediately after cell isolation (t = 0) or cultured in X-VIVO 15 complete medium alone or in the presence of EPO or EPO and SCF, with or without LY (50 μmol/L) for 3, 6, or 13 days before analysis by flow cytometry and counting of viable cells. (A) LY inhibits the EPO-induced or EPO- and SCF-induced formation of GPA+ erythroid progenitors. One experiment of three is shown. (B) Time course of the development of absolute number of viable GPA+ erythroid cells induced by EPO with or without LY or (C) EPO and SCF with or without LY. Mean ± SEM of three experiments. (D) Absolute number of viable GPA+ erythroid cells developed from CD34+CD71+CD45RA− erythroid progenitors stimulated with EPO with or without LY. Mean ± SEM of two experiments. Total number of viable cells in cultures stimulated with or without LY in the presence of (E) EPO or (F) EPO and SCF. Graphs show mean ± SEM of three experiments. (G) Total number of viable cells in cultures of CD34+CD71+CD45RA− erythroid progenitors stimulated with EPO with or without LY. Mean ± SEM of two experiments.
Experimental Hematology  , DOI: ( /S X(02) )

9. Figure 4
Time course of LY (LY)-mediated inhibition of EPO-induced or EPO- and SCF-induced formation of erythroid cells from CD34+ progenitor cells or from CD34+CD71+CD45RA− erythroid progenitors. CD34+ cells were immunophenotyped immediately after cell isolation (t = 0) or cultured in X-VIVO 15 complete medium alone or in the presence of EPO or EPO and SCF, with or without LY (50 μmol/L) for 3, 6, or 13 days before analysis by flow cytometry and counting of viable cells. (A) LY inhibits the EPO-induced or EPO- and SCF-induced formation of GPA+ erythroid progenitors. One experiment of three is shown. (B) Time course of the development of absolute number of viable GPA+ erythroid cells induced by EPO with or without LY or (C) EPO and SCF with or without LY. Mean ± SEM of three experiments. (D) Absolute number of viable GPA+ erythroid cells developed from CD34+CD71+CD45RA− erythroid progenitors stimulated with EPO with or without LY. Mean ± SEM of two experiments. Total number of viable cells in cultures stimulated with or without LY in the presence of (E) EPO or (F) EPO and SCF. Graphs show mean ± SEM of three experiments. (G) Total number of viable cells in cultures of CD34+CD71+CD45RA− erythroid progenitors stimulated with EPO with or without LY. Mean ± SEM of two experiments.
Experimental Hematology  , DOI: ( /S X(02) )

10. Figure 4
Time course of LY (LY)-mediated inhibition of EPO-induced or EPO- and SCF-induced formation of erythroid cells from CD34+ progenitor cells or from CD34+CD71+CD45RA− erythroid progenitors. CD34+ cells were immunophenotyped immediately after cell isolation (t = 0) or cultured in X-VIVO 15 complete medium alone or in the presence of EPO or EPO and SCF, with or without LY (50 μmol/L) for 3, 6, or 13 days before analysis by flow cytometry and counting of viable cells. (A) LY inhibits the EPO-induced or EPO- and SCF-induced formation of GPA+ erythroid progenitors. One experiment of three is shown. (B) Time course of the development of absolute number of viable GPA+ erythroid cells induced by EPO with or without LY or (C) EPO and SCF with or without LY. Mean ± SEM of three experiments. (D) Absolute number of viable GPA+ erythroid cells developed from CD34+CD71+CD45RA− erythroid progenitors stimulated with EPO with or without LY. Mean ± SEM of two experiments. Total number of viable cells in cultures stimulated with or without LY in the presence of (E) EPO or (F) EPO and SCF. Graphs show mean ± SEM of three experiments. (G) Total number of viable cells in cultures of CD34+CD71+CD45RA− erythroid progenitors stimulated with EPO with or without LY. Mean ± SEM of two experiments.
Experimental Hematology  , DOI: ( /S X(02) )

11. Figure 4
Time course of LY (LY)-mediated inhibition of EPO-induced or EPO- and SCF-induced formation of erythroid cells from CD34+ progenitor cells or from CD34+CD71+CD45RA− erythroid progenitors. CD34+ cells were immunophenotyped immediately after cell isolation (t = 0) or cultured in X-VIVO 15 complete medium alone or in the presence of EPO or EPO and SCF, with or without LY (50 μmol/L) for 3, 6, or 13 days before analysis by flow cytometry and counting of viable cells. (A) LY inhibits the EPO-induced or EPO- and SCF-induced formation of GPA+ erythroid progenitors. One experiment of three is shown. (B) Time course of the development of absolute number of viable GPA+ erythroid cells induced by EPO with or without LY or (C) EPO and SCF with or without LY. Mean ± SEM of three experiments. (D) Absolute number of viable GPA+ erythroid cells developed from CD34+CD71+CD45RA− erythroid progenitors stimulated with EPO with or without LY. Mean ± SEM of two experiments. Total number of viable cells in cultures stimulated with or without LY in the presence of (E) EPO or (F) EPO and SCF. Graphs show mean ± SEM of three experiments. (G) Total number of viable cells in cultures of CD34+CD71+CD45RA− erythroid progenitors stimulated with EPO with or without LY. Mean ± SEM of two experiments.
Experimental Hematology  , DOI: ( /S X(02) )

12. Figure 4
Time course of LY (LY)-mediated inhibition of EPO-induced or EPO- and SCF-induced formation of erythroid cells from CD34+ progenitor cells or from CD34+CD71+CD45RA− erythroid progenitors. CD34+ cells were immunophenotyped immediately after cell isolation (t = 0) or cultured in X-VIVO 15 complete medium alone or in the presence of EPO or EPO and SCF, with or without LY (50 μmol/L) for 3, 6, or 13 days before analysis by flow cytometry and counting of viable cells. (A) LY inhibits the EPO-induced or EPO- and SCF-induced formation of GPA+ erythroid progenitors. One experiment of three is shown. (B) Time course of the development of absolute number of viable GPA+ erythroid cells induced by EPO with or without LY or (C) EPO and SCF with or without LY. Mean ± SEM of three experiments. (D) Absolute number of viable GPA+ erythroid cells developed from CD34+CD71+CD45RA− erythroid progenitors stimulated with EPO with or without LY. Mean ± SEM of two experiments. Total number of viable cells in cultures stimulated with or without LY in the presence of (E) EPO or (F) EPO and SCF. Graphs show mean ± SEM of three experiments. (G) Total number of viable cells in cultures of CD34+CD71+CD45RA− erythroid progenitors stimulated with EPO with or without LY. Mean ± SEM of two experiments.
Experimental Hematology  , DOI: ( /S X(02) )

13. Figure 4
Time course of LY (LY)-mediated inhibition of EPO-induced or EPO- and SCF-induced formation of erythroid cells from CD34+ progenitor cells or from CD34+CD71+CD45RA− erythroid progenitors. CD34+ cells were immunophenotyped immediately after cell isolation (t = 0) or cultured in X-VIVO 15 complete medium alone or in the presence of EPO or EPO and SCF, with or without LY (50 μmol/L) for 3, 6, or 13 days before analysis by flow cytometry and counting of viable cells. (A) LY inhibits the EPO-induced or EPO- and SCF-induced formation of GPA+ erythroid progenitors. One experiment of three is shown. (B) Time course of the development of absolute number of viable GPA+ erythroid cells induced by EPO with or without LY or (C) EPO and SCF with or without LY. Mean ± SEM of three experiments. (D) Absolute number of viable GPA+ erythroid cells developed from CD34+CD71+CD45RA− erythroid progenitors stimulated with EPO with or without LY. Mean ± SEM of two experiments. Total number of viable cells in cultures stimulated with or without LY in the presence of (E) EPO or (F) EPO and SCF. Graphs show mean ± SEM of three experiments. (G) Total number of viable cells in cultures of CD34+CD71+CD45RA− erythroid progenitors stimulated with EPO with or without LY. Mean ± SEM of two experiments.
Experimental Hematology  , DOI: ( /S X(02) )

14. Figure 4
Time course of LY (LY)-mediated inhibition of EPO-induced or EPO- and SCF-induced formation of erythroid cells from CD34+ progenitor cells or from CD34+CD71+CD45RA− erythroid progenitors. CD34+ cells were immunophenotyped immediately after cell isolation (t = 0) or cultured in X-VIVO 15 complete medium alone or in the presence of EPO or EPO and SCF, with or without LY (50 μmol/L) for 3, 6, or 13 days before analysis by flow cytometry and counting of viable cells. (A) LY inhibits the EPO-induced or EPO- and SCF-induced formation of GPA+ erythroid progenitors. One experiment of three is shown. (B) Time course of the development of absolute number of viable GPA+ erythroid cells induced by EPO with or without LY or (C) EPO and SCF with or without LY. Mean ± SEM of three experiments. (D) Absolute number of viable GPA+ erythroid cells developed from CD34+CD71+CD45RA− erythroid progenitors stimulated with EPO with or without LY. Mean ± SEM of two experiments. Total number of viable cells in cultures stimulated with or without LY in the presence of (E) EPO or (F) EPO and SCF. Graphs show mean ± SEM of three experiments. (G) Total number of viable cells in cultures of CD34+CD71+CD45RA− erythroid progenitors stimulated with EPO with or without LY. Mean ± SEM of two experiments.
Experimental Hematology  , DOI: ( /S X(02) )

15. Figure 5
Effect of LY (LY) on survival of CD34+CD71+CD45RA− erythroid progenitors. CD34+CD71+CD45RA− FACS-sorted cells were cultured in X-VIVO 15 complete medium with the indicated cytokines in the presence or absence of LY (50 μmol/L). (A) Cells were cultured for 4 days and then stained with propidium iodide (PI). Data represent mean ± SEM of four separate experiments. (B) Cells were cultured for 4 days and then subjected to TUNEL analysis. Mean ± SEM of four separate experiments. *p < 0.020, EPO vs medium or EPO and LY.
Experimental Hematology  , DOI: ( /S X(02) )

16. Figure 5
Effect of LY (LY) on survival of CD34+CD71+CD45RA− erythroid progenitors. CD34+CD71+CD45RA− FACS-sorted cells were cultured in X-VIVO 15 complete medium with the indicated cytokines in the presence or absence of LY (50 μmol/L). (A) Cells were cultured for 4 days and then stained with propidium iodide (PI). Data represent mean ± SEM of four separate experiments. (B) Cells were cultured for 4 days and then subjected to TUNEL analysis. Mean ± SEM of four separate experiments. *p < 0.020, EPO vs medium or EPO and LY.
Experimental Hematology  , DOI: ( /S X(02) )

17. Figure 6
LY (LY) inhibits EPO-induced erythroid maturation but has less effect when EPO is combined with SCF. CD34+GPA−CFSEmean FACS-sorted cells were cultured in X-VIVO 15 complete medium with the indicated cytokines in the presence or absence of LY (50 μmol/L) for different time periods. The cells then were stained with anti–IgG1-PE or anti–GPA-PE and analyzed by flow cytometry to determine their proliferation history in relation to their differentiation status. In each dot plot, the number of cell divisions is indicated. One representative experiment of four is shown.
Experimental Hematology  , DOI: ( /S X(02) )

18. Figure 7
EPO-induced activation of Akt is suppressed by various inhibitors of PKC. CD34+ cells were cultured in X-VIVO 15 complete medium overnight, followed by incubation with staurosporine (20 nmol/L), calphostin C (Cal C; 50 nmol/L), or Gö6976 (125 nmol/L) for 20 minutes before stimulation with EPO (10 U/mL) for another 20 minutes. Cells were lysed and 75% of the cell lysate was immunoprecipitated with anti-Akt antibody and the immunoprecipitates subjected to an in vitro kinase assay using GSK-3 fusion protein as a substrate (1 μg). Reaction products were resolved by 12% SDS-PAGE and subjected to immunoblot analysis using anti-phospho–GSK-3 antibody. The remaining 25% of cell lysate were run on 12% SDS-PAGE and hybridized with actin Ab. (A) One representative experiment of three is shown. (B) Relative expression of phospho–GSK-3 α/β determined by densitometric scanning of hyperfilms. Mean ± SEM of three experiments.
Experimental Hematology  , DOI: ( /S X(02) )

19. Figure 7
EPO-induced activation of Akt is suppressed by various inhibitors of PKC. CD34+ cells were cultured in X-VIVO 15 complete medium overnight, followed by incubation with staurosporine (20 nmol/L), calphostin C (Cal C; 50 nmol/L), or Gö6976 (125 nmol/L) for 20 minutes before stimulation with EPO (10 U/mL) for another 20 minutes. Cells were lysed and 75% of the cell lysate was immunoprecipitated with anti-Akt antibody and the immunoprecipitates subjected to an in vitro kinase assay using GSK-3 fusion protein as a substrate (1 μg). Reaction products were resolved by 12% SDS-PAGE and subjected to immunoblot analysis using anti-phospho–GSK-3 antibody. The remaining 25% of cell lysate were run on 12% SDS-PAGE and hybridized with actin Ab. (A) One representative experiment of three is shown. (B) Relative expression of phospho–GSK-3 α/β determined by densitometric scanning of hyperfilms. Mean ± SEM of three experiments.
Experimental Hematology  , DOI: ( /S X(02) )