1. Enhancement of intracellular signaling associated with hematopoietic progenitor cell survival in response to SDF-1/CXCL12 in synergy with other cytokines
by Younghee Lee, Akihiko Gotoh, Hyung-Joo Kwon, Minute You, Lisa Kohli, Charlie Mantel, Scott Cooper, Giao Hangoc, Keisuke Miyazawa, Kazuma Ohyashiki, and Hal E. Broxmeyer
Blood
Volume 99(12):
June 15, 2002
©2002 by American Society of Hematology

2. Activation of Akt, ERK1/2, and p90RSK in MO7e cells after stimulation with SDF-1α.(A) MO7e cells were incubated with 100 ng/mL SDF-1α for the indicated time periods.
Activation of Akt, ERK1/2, and p90RSK in MO7e cells after stimulation with SDF-1α.(A) MO7e cells were incubated with 100 ng/mL SDF-1α for the indicated time periods. (B) MO7e cells were preincubated in the presence of dimethyl sulfoxide (DM) vehicle control or LY (LY, 30 μM), wortmannin (W, 100 nM), PD (PD, 25 μM), or rapamycin (R [also called sirolimus], 10 nM) for 1 hour, and stimulated with 100 ng/mL SDF-1α for 5 minutes. Cell lysates were analyzed by Western blotting with phosphospecific antibodies to Akt (Ser473), ERK1/2 (Thr202/Tyr204), or p90RSK (Ser381). Amounts of p90RSK (A) or Akt (B) are shown on the bottom panel of each as a loading control. This experiment was performed 3 times with similar results.
Younghee Lee et al. Blood 2002;99:
©2002 by American Society of Hematology

3. Synergistic activation of ERK1/2 and p90RSK by SDF-1α in combination with other cytokines.MO7e cells were incubated for the indicated time periods with SDF-1α (100 ng/mL), GM-CSF (10 ng/mL; GM), SLF (50 ng/mL), or TPO (50 ng/mL) each alone, or with the comb...
Synergistic activation of ERK1/2 and p90RSK by SDF-1α in combination with other cytokines.MO7e cells were incubated for the indicated time periods with SDF-1α (100 ng/mL), GM-CSF (10 ng/mL; GM), SLF (50 ng/mL), or TPO (50 ng/mL) each alone, or with the combination of SDF-1α plus one of these cytokines. Cell lysates were analyzed by Western blotting with phosphospecific antibodies to ERK1/2 (Thr202/Tyr204) or p90RSK (Ser381). The amount of p90RSK is shown as a loading control in the bottom panels. (A) SDF-1α plus GM-CSF. (B) SDF-1α plus SLF. (C) SDF-1α plus TPO. (D,E) Columns represent relative band intensities of phospho-ERK (D) and phospho-RSK (E) ± SD from 3 experiments. *P < .05 (greater than additive).
Younghee Lee et al. Blood 2002;99:
©2002 by American Society of Hematology

4. Synergistic activation of Akt in MO7e cells by SDF-1α in combination with other cytokines.Cell lysates were analyzed by Western blotting with phosphospecific antibodies to Akt (Ser473).
Synergistic activation of Akt in MO7e cells by SDF-1α in combination with other cytokines.Cell lysates were analyzed by Western blotting with phosphospecific antibodies to Akt (Ser473). The amount of total Akt is shown as a loading control in the bottom panels. (A) SDF-1α plus GM-CSF. (B) SDF-1α plus SLF. (C) SDF-1α plus TPO. (D) Columns represent relative band intensities of phospho-Akt ± SD from 3 experiments. *P < .05 (greater than additive).
Younghee Lee et al. Blood 2002;99:
©2002 by American Society of Hematology

5. Synergistic activation of ERK1/2 by SDF-1α in combination with nonproliferative concentrations of other cytokines.MO7e cells were stimulated for 15 minutes with SDF-1α alone, another cytokine alone, or SDF-1α plus another cytokine at the indicated concentra...
Synergistic activation of ERK1/2 by SDF-1α in combination with nonproliferative concentrations of other cytokines.MO7e cells were stimulated for 15 minutes with SDF-1α alone, another cytokine alone, or SDF-1α plus another cytokine at the indicated concentrations. Cell lysates were analyzed by Western blotting using phosphospecific antibodies to ERK1/2 (Thr202/Tyr204). The membrane was reblotted with antibody recognizing p90RSK to show equal loading. (A) SDF-1α plus GM-CSF. (B) SDF-1α plus SLF. (C) SDF-1α plus TPO. (D) Columns represent relative band intensities of phospho-ERK ± SD from 3 experiments with similar results. *P < .05 (greater than additive).
Younghee Lee et al. Blood 2002;99:
©2002 by American Society of Hematology

6. Enhanced activation of MAPK and Akt signaling by pretreatment of MO7e cells with SDF-1α.Factor-starved MO7e cells were preincubated with or without 100 ng/mL of either SDF-1α, MIP-1α, or RANTES for 30 minutes or the indicated periods of time (Aiii), followe...
Enhanced activation of MAPK and Akt signaling by pretreatment of MO7e cells with SDF-1α.Factor-starved MO7e cells were preincubated with or without 100 ng/mL of either SDF-1α, MIP-1α, or RANTES for 30 minutes or the indicated periods of time (Aiii), followed by treatment with 10 ng/mL SLF or 1 ng/mL GM-CSF for 5 minutes. In some experiments, cells were pretreated with PD98059 for 1 hour before SLF treatment (Aiv). (A) ERK immunoprecipitates obtained from total cell lysate using anti–phospho-ERK mAb were subjected to MAPK activity assay as described in “Materials and methods.” Recombinant active MAPK (20 ng) was included as a positive control (Ai). These results are representative of 3 independent experiments. (B) Phosphorylation levels of Akt were determined by Western blotting with phosphospecific antibodies to Akt (Ser473). The amount of total Akt is shown as a loading control in the bottom panels of B. This is a representative of 3 separate experiments with similar results.
Younghee Lee et al. Blood 2002;99:
©2002 by American Society of Hematology

7. No effect of SDF-1α or other cytokines on translocation of NF-κB
No effect of SDF-1α or other cytokines on translocation of NF-κB.Factor-starved MO7e cells were stimulated with SDF-1α alone, other cytokines alone, or combinations of cytokines for 15 minutes, and the cellular localization of NF-κB was determined by confoc...
No effect of SDF-1α or other cytokines on translocation of NF-κB.Factor-starved MO7e cells were stimulated with SDF-1α alone, other cytokines alone, or combinations of cytokines for 15 minutes, and the cellular localization of NF-κB was determined by confocal microscopy after staining with a mAb against human NF-κB p65. Nuclei were stained by Hoechst no Cells treated with TNF-α (15 minutes) are shown as a positive control for NF-κB nuclear translocation. Magnification, × 100.
Younghee Lee et al. Blood 2002;99:
©2002 by American Society of Hematology

8. Influence of SDF-1α, GM-CSF, TPO, SLF, and FL, alone and in combination on the survival of CFU-GMs and CFU-GEMMs in FACS-sorted CD34+++ cord blood cells subjected to delayed addition of a combination of maximally stimulating growth factors.Human CD34+++ cel...
Influence of SDF-1α, GM-CSF, TPO, SLF, and FL, alone and in combination on the survival of CFU-GMs and CFU-GEMMs in FACS-sorted CD34+++ cord blood cells subjected to delayed addition of a combination of maximally stimulating growth factors.Human CD34+++ cells were plated at time 0 in the absence and presence of various concentrations of SDF-1α, GM-CSF, TPO, SLF, FL, or SDF-1α plus either GM-CSF, TPO, SLF, or FL. The combination of rhu Epo (1 U/mL), rhu GM-CSF (10 ng/mL), rhu IL-3 (10 ng/mL), and rhu SLF (50 ng/mL), a maximally potent combination of cytokines to stimulate colony formation, was added to the plates at either time 0 or 24 hours and cultures were scored for CFU-GM and CFU-GEMM colonies 14 days after the addition of the maximally stimulating cytokines. Results are given as colonies ± SD (with the percent survival given in parentheses). (A) Significant increase in survival compared to control plates at the same time of delayed growth factor addition (P < .001). (B) Significant increase in survival compared to control plates at same time of delayed growth factor addition (P < .01). (C) Significant increase in survival compared to control plates at same time of delayed growth factor additions (P < .05). (D) Significantly greater survival than either cytokine alone and additive to slightly less than additive effects at the same time of delayed growth factor addition (P < .05). (E) Significantly greater survival than with either cytokine alone and greater than additive effect at the same time of delayed growth factor addition (P < .01). The addition of either SDF-1, GM-CSF, TPO, SLF, FL, or the combination of SDF-1 plus these cytokines along with the maximally stimulating combination of Epo, GM-CSF, IL-3, and SLF at time 0 had no significant effect (P > .05) compared to control medium added with the maximally stimulating combination of growth factors at time 0 (data not shown).
Younghee Lee et al. Blood 2002;99:
©2002 by American Society of Hematology

9. Influence of SDF-1α, GM-CSF, TPO, and SLF on survival of MO7e colony-forming cells (CFCs) after growth factor withdrawal.MO7e cells were plated at 103 cells/mL in agar culture medium at time 0 with control medium or the amounts of SDF-1α, GM-CSF, TPO, or SL...
Influence of SDF-1α, GM-CSF, TPO, and SLF on survival of MO7e colony-forming cells (CFCs) after growth factor withdrawal.MO7e cells were plated at 103 cells/mL in agar culture medium at time 0 with control medium or the amounts of SDF-1α, GM-CSF, TPO, or SLF, or SDF-1α plus either GM-CSF, TPO, or SLF shown. The combination of GM-CSF (10 ng/mL) plus SLF (50 ng/mL), a maximal concentration of factors to stimulate MO7e colony formation, was added to these plates at either time 24 or 48 hours. Results are shown as colonies ± SD (with the percent survival compared to time 0 culture plated with control medium shown in parentheses). Statistical differences between groups are noted by the same letters as for the results in Figure 7. The addition of either SDF-1, GM-CSF, TPO, SLF, or SDF-1 plus these cytokines along with the maximally stimulating combination of GM-CSF plus SLF at time 0 had no significant effect (P > .05) compared to control medium added with the maximally stimulating combination of growth factors at time 0 (data not shown).
Younghee Lee et al. Blood 2002;99:
©2002 by American Society of Hematology

10. Effect of SDF-1α on apoptosis induced by growth factor withdrawal
Effect of SDF-1α on apoptosis induced by growth factor withdrawal.Factor-starved CD34+ cells prepared from human bone marrow (A) or MO7e cells (B) were further incubated in serum-free media in the presence of either 100 ng/mL SDF-1α alone, 10 ng/mL SLF alon...
Effect of SDF-1α on apoptosis induced by growth factor withdrawal.Factor-starved CD34+ cells prepared from human bone marrow (A) or MO7e cells (B) were further incubated in serum-free media in the presence of either 100 ng/mL SDF-1α alone, 10 ng/mL SLF alone, or the combination of these 2 cytokines. After 4 days, cells were stained with PC-5–conjugated APO2.7 mAb and analyzed by flow cytometry. (A) Values in histogram represent percent of APO2.7+ cells. This is representative of 3 separate experiments. (B) Columns represent the average percent of APO2.7+ cells ± SD of 3 separate experiments. *P < .01 versus SLF alone.
Younghee Lee et al. Blood 2002;99:
©2002 by American Society of Hematology