Reexposure of AML cells adjusted to 6% O2 result in a very rapid loss of surface and total CXCR4. (A) Live imaging (40×/1.5 NA) of OCI-AML3 cells at 6% (left) and 5 minutes reoxygenation with 21% O2 (right) show the loss of bound FITC-labeled CXCR4 antibody. (B) Similar results are found for U937 cells. (C) Flow cytometric analysis of OCI-AML3, either surface-stained or fixed with 2% PFA and methanol permeabilized for total CXCR4, shows a simultaneous loss of surface CXCR4 (left, ○) and total protein (right, ▴) within 5 minutes. Graphs show means and SD. (D) Treatment of OCI-AML3 with proteasome inhibitor lactacystin (10 mM) before reoxygenation prevents neither loss of surface expression of CXCR4 by flow cytometry nor total CXCR4 by Western blotting.
