Treatment with PS depletes CXCR4 mRNA and protein expression in cultured and primary AML cells. (A) OCI-AML3 and HL-60 cells were treated with the indicated concentrations of PS for 16 hours. Then, total RNA was extracted and RT-PCR was performed for CXCR4, p21, and p27. A β-actin–specific PCR reaction served as a loading control for the amplification. (B) OCI-AML3 and HL-60 cells were treated with the indicated concentration of PS for 24 hours. After treatment, immunoblot analyses were performed for CXCR4 and p21 on the total cell lysates. The expression of β-actin in the lysates served as the loading control. (C) OCI-AML3 and HL-60 cells were treated with 50nM PS for the indicated times, and immunoblot analyses were performed as described in panel B. (D) OCI-AML3 and primary AML cells were treated with the indicated concentrations of PS for 24 hours. Then surface expression of CXCR4 was analyzed by flow cytometry using monoclonal phycoerythrin-conjugated 12G5 antibody. Values indicate the relative fluorescence of CXCR4 detected. (E) OCI-AML3 cells were treated with the indicated concentrations of PS for 4 and 16 hours. After treatment, total RNA was isolated and reverse transcribed with a specific stem loop primer for hsa-miR-146a. The resulting cDNAs were used for quantitative PCR with a TaqMan probe for hsa-miR-146a. Relative expression levels were normalized against 18S rRNA. (F) OCI-AML3 cells were treated with the indicated concentrations of PS for 24 hours in the presence of cobalt chloride. After treatment, cell lysates were prepared and immunoblot analyses were performed for HIF-1α, CXCR4, and hsp70. The expression levels of β-actin in the lysates served as the loading control.