Effect of hypoxia on MM cell homing to the BM in vivo. (A) The effect of incubation of MM1s cells under hypoxic conditions for 24 hours in vitro on homing to the BM of mice after IV injection detected by in vivo flow cytometry. This showed that hypoxia accelerated the homing of MM cells to the BM. (B) Results of homing of MM1s cells with HIF1α knockdown incubated in hypoxic or normoxic conditions for 24 hours before injection into the tail veins of mice. Hypoxia did not accelerate homing of MM cells with knockdown of HIF1α. (C) Pretreatment of hypoxic and normoxic cells with the CXCR4 inhibitor reversed their homing. MM cells were incubated under hypoxic or normoxic conditions for 24 hours and injected into mice. (D) Homing of MM cells to the BM of mouse skulls was imaged 5-7 minutes after the injection using live in vivo confocal microscopy. This shows that hypoxia increased the number of MM cells that homed to the BM, shown as the mean number of MM cells in each frame or as representative images showing the increase in the number of hypoxic cells in BM niches compared with normoxic cells (E). Green indicates MM cells; red, blood vessels.