HIF1α transcriptional activity is necessary for PGE₂-induced CXCR4 upregulation. (A) (Left) CXCR4 cell surface expression (X ± SEM; N = 3 experiments) on HIF1β (−) and HIF1β (+) cells 24 hours after treatment with dmPGE₂. CXCR4 cell surface expression was determined by flow cytometry. Data are expressed as percent change in mean fluorescence intensity of CXCR4 over vehicle. *P < .05. (Right) CXCR4 cell surface expression (X ± SEM; N = 3 experiments) in HIF1β (−) and HIF1β (+) cells 2 hours after treatment with vehicle or dmPGE₂ determined by quantitative reverse-transcription polymerase chain reaction. (B) Schematic of pGL2b luciferase reporter constructs containing various regions of the murine CXCR4 promoter. (C) In vitro Luciferase reporter assay. 293T cells were transfected with either full-length CXCR4 promoter constructs containing all HREs (Full), truncated constructs containing 1 HRE (ΔHRE1) or 2 HREs (ΔHRE2), or a mutated 1.3 kb of HRE (HRE 2 Mut). After 24 hours, cells were split equally and treated with either vehicle or dmPGE₂ for 16 hours at 37°C. Luciferase activity was measured using the Firefly Luciferase assay kit (Promega). Data are represented as X ± SEM from 2 separate experiments (N = 6). *P < .05. (D) (Top) Representative blot of HIF1α protein 4 hours after treatment with vehicle, dmPGE₂, or DMOG, with or without the addition of SNP. (Bottom) Densitometry analysis of HIF1α protein expression in mouse lineage^neg BMCs treated with vehicle, dmPGE₂, or DMOG, with or without the addition of SNP. Data are expressed as X ± standard deviation percent change in protein levels over vehicle control from 2 separate experiments. (E) Expression of HIF1 responsive genes after treatment with vehicle or dmPGE₂ with or without SNP. Data are expressed as X ± SEM, N = 3 experiments. (F) (Left) In vitro transwell migration of murine SKL cells to 100 ng/mL of SDF-1. Lineage^neg cells were treated with vehicle or 1 µM of dmPGE₂ with or without 100 µM of SNP for 2 hours at 37°C. Data are expressed as mean ± SEM, N = 3 experiments. *P < .05. (Right) Representative fluorescence-activated cell sorter histogram showing CXCR4 expression on SKL cells compared with isotype control. (G) In vivo homing of HIF1α KO cells. BMCs from conditional HIF1α KO or floxed control (CD45.2) mice were treated with vehicle or dmPGE₂, and 1 × 10⁶ treated lineage^neg cells were transplanted into lethally irradiated BoyJ (CD45.1) mice. At 16 hours later, BM was analyzed for homed SKL cells. Data are represented as X ± SEM from 1 experiment (N = 4-5 mice/group/experiment, each assayed individually). (H) CXCR4 expression (X ± SEM) on HIF1α KO and HIF1α Floxed SKL cells after treatment with dmPGE₂. N = 3 mice/group, each assayed individually. *P < .05.