American Society of Hematology

Figure 1

PGE2increases HIF1α protein and downstream responsive genes. (A) (Top) Representative blot of HIF1α protein 4 hours after treatment with vehicle, 1 µM of dmPGE2, or 5 µM of DMOG. (Bottom) Densitometry analysis of HIF1α protein expression in mouse lineageneg BMCs treated with vehicle, dmPGE2, or DMOG. Data are expressed as X ± standard error of the mean (SEM) percent change in protein levels over vehicle control from 3 separate experiments. (B) Expression of HIF1 responsive genes in mouse lineageneg BMCs after treatment with dmPGE2 and DMOG as determined by quantitative reverse-transcription polymerase chain reaction. Data are X ± SEM, N = 3 experiments. *P < .05. (C) (Top) Dose-response blot of HIF1α protein 4 hours after treatment with vehicle or DMOG. (Bottom) Densitometry analysis of HIF1α protein expression in mouse lineageneg BMC. (D) Dose response of CXCR4 on SKL cells treated with vehicle or DMOG. Lineageneg BMCs were treated with DMOG for 2 hours at 37°C, washed, and incubated in RPMI with 10% heat-inactivated fetal calf serum for 16 hours. CXCR4 expression on LSK-gated cells was analyzed by flow cytometry. (E) In vitro transwell migration of murine SKL cells. One million lineageneg BMCs were treated with vehicle, dmPGE2, or DMOG. Cells were assayed for the ability to migrate to 100 ng/mL recombinant murine SDF-1 for 4 hours at 37°C. Data are expressed as X ± SEM, N = 9. (F) BMCs from CD45.1 mice were treated with vehicle, 1 µM of dmPGE2, or 5 µM of DMOG and 1 × 106 treated lineageneg cells transplanted into lethally irradiated CD45.2 mice. At 16 hours later, BM was analyzed for homed SKL cells. Data are represented as X ± SEM from 2 separate experiments (N = 4-5 mice/group/experiment, each assayed individually). (G) Similar homing experiment with or without the addition of AMD3100 10 minutes before transplantation. Data are represented as X ± SEM from 2 separate experiments (N = 4-5 mice/group/experiment, each assayed individually) *P < .05. (H) Percent contribution (chimerism) and (I) frequency analysis for DMOG determined by Poisson statistics using LCALC software. Vehicle P0= 121 319 and DMOG P0= 53 956. (J) Competitive repopulating units of DMOG and vehicle-treated cells in peripheral blood 6 months after transplant. Data are represented as X ± SEM from 2 pooled experiments (N = 5 mice/group/experiment, each assayed individually). (K) Secondary transplant percent chimerism at 6 months after transplant (N = 6 mice/group each assayed individually). *P < .05.

PGE₂increases HIF1α protein and downstream responsive genes. (A) (Top) Representative blot of HIF1α protein 4 hours after treatment with vehicle, 1 µM of dmPGE₂, or 5 µM of DMOG. (Bottom) Densitometry analysis of HIF1α protein expression in mouse lineage^neg BMCs treated with vehicle, dmPGE₂, or DMOG. Data are expressed as X ± standard error of the mean (SEM) percent change in protein levels over vehicle control from 3 separate experiments. (B) Expression of HIF1 responsive genes in mouse lineage^neg BMCs after treatment with dmPGE₂ and DMOG as determined by quantitative reverse-transcription polymerase chain reaction. Data are X ± SEM, N = 3 experiments. *P < .05. (C) (Top) Dose-response blot of HIF1α protein 4 hours after treatment with vehicle or DMOG. (Bottom) Densitometry analysis of HIF1α protein expression in mouse lineage^neg BMC. (D) Dose response of CXCR4 on SKL cells treated with vehicle or DMOG. Lineage^neg BMCs were treated with DMOG for 2 hours at 37°C, washed, and incubated in RPMI with 10% heat-inactivated fetal calf serum for 16 hours. CXCR4 expression on LSK-gated cells was analyzed by flow cytometry. (E) In vitro transwell migration of murine SKL cells. One million lineage^neg BMCs were treated with vehicle, dmPGE₂, or DMOG. Cells were assayed for the ability to migrate to 100 ng/mL recombinant murine SDF-1 for 4 hours at 37°C. Data are expressed as X ± SEM, N = 9. (F) BMCs from CD45.1 mice were treated with vehicle, 1 µM of dmPGE₂, or 5 µM of DMOG and 1 × 10⁶ treated lineage^neg cells transplanted into lethally irradiated CD45.2 mice. At 16 hours later, BM was analyzed for homed SKL cells. Data are represented as X ± SEM from 2 separate experiments (N = 4-5 mice/group/experiment, each assayed individually). (G) Similar homing experiment with or without the addition of AMD3100 10 minutes before transplantation. Data are represented as X ± SEM from 2 separate experiments (N = 4-5 mice/group/experiment, each assayed individually) *P < .05. (H) Percent contribution (chimerism) and (I) frequency analysis for DMOG determined by Poisson statistics using LCALC software. Vehicle P₀= 121 319 and DMOG P₀= 53 956. (J) Competitive repopulating units of DMOG and vehicle-treated cells in peripheral blood 6 months after transplant. Data are represented as X ± SEM from 2 pooled experiments (N = 5 mice/group/experiment, each assayed individually). (K) Secondary transplant percent chimerism at 6 months after transplant (N = 6 mice/group each assayed individually). *P < .05.

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