Suppression of CD63 in HL-60 cells does not affect the level of proNE but diminishes the amount of mature NE and inhibits proNE processing and secretion. (A) CD63 of HL-60 cells is suppressed by CD63 siRNA—real-time PCR. The figure shows the relative mRNA levels for CD63 compared with wild-type cells (wt) with 18S mRNA as an internal control. The clones shown are CD63 siRNA mock, CD63 siRNA rev (reverted CD63 siRNA clone), CD63 siRNA (5 clones), CD63 dom neg mock, CD63 dom neg rev (reverted CD63 dom neg clone), and CD63 dom neg (2 clones). Data presented are mean plus or minus SD of 1 representative experiment of 3 similar experiments, each done in triplicate from 1 run of real-time PCR, and calculated according to the ΔΔC_T method. The mean suppression of all CD63 siRNA clones was 6.5-fold for CD63. CD63 dom neg clones showed no significant suppression of CD63 mRNA. Notice the normalization of CD63 mRNA levels in the reverted CD63 siRNA clone. (B) ProNE of HL-60 cells is not diminished by CD63 depletion—Western blotting. The panel shows immunoblotting with anti-proNE, anti-MPO, and anti-actin of cell lysates from the same clones as in panel A, except for lack of clone CD63 dom neg C1. The numbers on top represent the relative densitometry values normalized for wild-type (wt) cells. The amount of proNE detected in 4 of 5 different siRNA clones and 1 CD63 dom neg clone was similar to wild-type cells. Actin is shown as control. Molecular weights in kilodaltons are shown to the left. (C) NE processing and secretion are inhibited—biosynthetic radiolabeling. Wild-type (wt), CD63 siRNA mock (siRNA mock), CD63 siRNA (siRNA), and CD63 dom neg (dom neg) HL-60 cells were radiolabeled (pulse) for 30 minutes, followed by chase of the radiolabel for up to 4 hours. The 85-kDa band of the supernatant corresponds to a covalent complex between proNE and α1-proteinase inhibitor. Different processing forms are seen in the figure: 31-kDa unglycosylated apoproNE, glycosylated 34-kDa proNE, and mature 29-kDa NE (marked with arrows). Wild-type and CD63 siRNA mock cells display the processing into mature NE forms and constitutive secretion of proNE. CD63 siRNA and CD63 dom neg cells lack both processing of proNE and constitutive secretion. The numbers to the left are molecular mass standards. (D) Processing and secretion of MPO in wild-type (wt), CD63 siRNA (siRNA), CD63 dom neg (dom neg), and CD63 siRNA mock-transfected (siRNA mock) cells was investigated by overnight radiolabeling and IP with anti-MPO and then examined by SDS-PAGE followed by fluorography. Processing of pro-MPO into mature α- and β-subunits was also observed after CD63 suppression by CD63 siRNA or CD63 dom neg. (E) NE of HL-60 cells is diminished by CD63 depletion—immunofluorescence microscopy. The panel shows CD63 and NE staining (using anti-NE antibody) observed in CD63 siRNA, CD63 siRNA mock, CD63 dom neg, and CD63 dom neg mock HL-60 cells. Depletion of CD63 by siRNA or a dom neg mutant was associated with down-regulation of NE.