NE of HL-60 cells is diminished by CD63 depletion—flow cytometry. Wild-type (wt), CD63 siRNA, CD63 siRNA mock (mock; thin line), reverted CD63 siRNA clone (siRNA rev; thick line), CD63 dom neg, CD63 dom neg mock (mock; thin line), and reverted CD63 dom neg (dom neg rev; thick line) HL-60 cells were incubated with anti-CD63 or anti-NE as primary antibody, then labeled with Alexa Fluor 488 as secondary antibody. When cells were stained on the surface only (nonpermeabilized; to the left), CD63 staining (upper panel) in CD63 siRNA cells decreased compared with wild-type and mock cells, but the levels in dom neg CD63–transfected cells were similar to wild-type and mock cells. The surface staining of NE (lower panel) was lower in both CD63 siRNA and CD63 dom neg cells than in wild-type and mock cells. After 8 weeks in culture, the clones reverted and regained their expression of CD63 and NE (although to a lesser degree), resembling mock and wild-type cells. Cells were also fixed and permeabilized to demonstrate the intracellular content of CD63 and NE (panels to the right). Regarding both CD63 and NE, the intracellular fluorescence of CD63 siRNA and CD63 dom neg cells was lower than that of wild-type and mock cells. Revertants recovered expression up to or equal to the same level as wild-type and mock cells. The plots are representative of 2 to 3 experiments.