Figure 5
Figure 5. CXCR4 receptor expression is increased on murine and human HSPCs after dmPGE2 treatment. (A) CXCR4 expression (mean ± SEM; n = 3) on murine KL and SKL cells, and human CD34+ UCB cells 24 hours after treatment with dmPGE2. Data are expressed as percentage change in mean fluorescence intensity (MFI) of CXCR4 due to treatment with dmPGE2 or vehicle. (B) Freshly isolated Linneg cells were pulsed with dmPGE2 or vehicle for 2 hours, washed, and resuspended in media with 10% HI-FCS and cultured at 37°C for 16 hours. After incubation, cells were washed, resuspended in RPMI/0.5% BSA, and allowed to migrate to rmSDF-1α for 4 hours. Total cell migration was quantitated by flow cytometry. Data are the mean plus or minus SEM percentage migration for 3 experiments. (†P < .05 for dmPGE2-treated cells compared with cells treated with vehicle.) (Top inset) Percentage migration of gated SKL cells to positive (100 ng/mL SDF-1 in bottom chamber), negative (100 ng/mL SDF1 in upper chamber), or neutral (100 ng/mL SDF-1 in both upper and bottom chambers) gradients. Data are the mean ± SEM percentage migration for 3 experiments. (†P < .05 for dmPGE2-treated cells compared with cells treated with vehicle.) (Bottom inset) Percentage migration of sorted SKL cells to 100 ng/mL SDF-1. Data are the mean plus or minus SEM percentage migration for 3 experiments. (†P < .05 for dmPGE2-treated cells compared with cells treated with vehicle.) (C) Freshly isolated UCB CD34+ cells were pulsed with dmPGE2 or vehicle for 2 hours, washed, and resuspended in media with 10% HI-FCS and cultured at 37°C for 16 hours. After incubation, cells were washed and resuspended in RPMI/0.5% BSA, and migration to rhSDF-1 was quantitated by flow cytometry. To block the CXCR4 receptor, replicate cells were incubated with 5 μg/mL AMD3100 for 30 minutes prior to the migration assay. Data are the mean plus or minus SEM percentage migration for 3 experiments. (†P < .05 for dmPGE2-treated cells compared with cells treated with vehicle.) (D) Homing efficiency of vehicle- and dmPGE2-treated cells to bone marrow in the absence and presence of 10 μM AMD3100. Cells were incubated with AMD3100 for 30 minutes prior to the homing assay. Data are expressed as mean plus or minus SEM; n = 3 mice/group, each assayed individually.

CXCR4 receptor expression is increased on murine and human HSPCs after dmPGE2 treatment. (A) CXCR4 expression (mean ± SEM; n = 3) on murine KL and SKL cells, and human CD34+ UCB cells 24 hours after treatment with dmPGE2. Data are expressed as percentage change in mean fluorescence intensity (MFI) of CXCR4 due to treatment with dmPGE2 or vehicle. (B) Freshly isolated Linneg cells were pulsed with dmPGE2 or vehicle for 2 hours, washed, and resuspended in media with 10% HI-FCS and cultured at 37°C for 16 hours. After incubation, cells were washed, resuspended in RPMI/0.5% BSA, and allowed to migrate to rmSDF-1α for 4 hours. Total cell migration was quantitated by flow cytometry. Data are the mean plus or minus SEM percentage migration for 3 experiments. (†P < .05 for dmPGE2-treated cells compared with cells treated with vehicle.) (Top inset) Percentage migration of gated SKL cells to positive (100 ng/mL SDF-1 in bottom chamber), negative (100 ng/mL SDF1 in upper chamber), or neutral (100 ng/mL SDF-1 in both upper and bottom chambers) gradients. Data are the mean ± SEM percentage migration for 3 experiments. (†P < .05 for dmPGE2-treated cells compared with cells treated with vehicle.) (Bottom inset) Percentage migration of sorted SKL cells to 100 ng/mL SDF-1. Data are the mean plus or minus SEM percentage migration for 3 experiments. (†P < .05 for dmPGE2-treated cells compared with cells treated with vehicle.) (C) Freshly isolated UCB CD34+ cells were pulsed with dmPGE2 or vehicle for 2 hours, washed, and resuspended in media with 10% HI-FCS and cultured at 37°C for 16 hours. After incubation, cells were washed and resuspended in RPMI/0.5% BSA, and migration to rhSDF-1 was quantitated by flow cytometry. To block the CXCR4 receptor, replicate cells were incubated with 5 μg/mL AMD3100 for 30 minutes prior to the migration assay. Data are the mean plus or minus SEM percentage migration for 3 experiments. (†P < .05 for dmPGE2-treated cells compared with cells treated with vehicle.) (D) Homing efficiency of vehicle- and dmPGE2-treated cells to bone marrow in the absence and presence of 10 μM AMD3100. Cells were incubated with AMD3100 for 30 minutes prior to the homing assay. Data are expressed as mean plus or minus SEM; n = 3 mice/group, each assayed individually.

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