Effect of hypoxia on the expression of CXCR4 and chemotaxis of MM cells. (A) MM cells isolated from the BM of mice injected with MM1s cells at different tumor burden were analyzed for the expression of CXCR4 by flow cytometry and compared with the hypoxic state in these cells as shown by the MFI of PIM in the MM cells. This shows that the expression of CXCR4 increased significantly with the increase in hypoxia in MM cells. (B) Expression of CXCR4 in MM cells isolated from the PB and BM of mice injected with MM1s cells, which shows a higher expression of CXCR4 in circulating MM cells compared with MM cells residing in the BM. Shown is the effect of incubation of MM cells (MM1s and H929) under hypoxic conditions for 24 hours in vitro on the expression of CXCR4 in MM cells (MM1s, H929, U266, and 5T33MMvt) detected at the protein level by flow cytometry (C) or detected mRNA level by quantitative RT-PCR (D). This shows that hypoxia increased the expression of CXCR4 in MM cells. (E) Effect of incubation of MM cells (MM1s and H929) under hypoxic conditions for 24 hours in vitro on chemotaxis toward SDF1α and (F) actin polymerization showing a significant increase of chemotaxis and actin polymerization in hypoxic MM cells. (G) Effect of the CXCR4 inhibitor AMD1300 on chemotaxis of hypoxic MM cells. It can be seen that chemotaxis was abolished. (H) Knockdown of HIF1α in MM1s cells showing decreased expression of HIF1α under hypoxic conditions in cells transfected with HIF1α siRNA. (I) Effect of knockdown of HIF1α on the expression of CXCR4 induced by hypoxia. This shows that knockdown of HIF1α decreased the expression of CXCR4 induced by hypoxia. (J) The effect of knockdown of HIF1α on the increased chemotaxis induced by hypoxia. This shows that knockdown of HIF1α reversed the increase of chemotaxis in response to hypoxia.