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Blood, Vol. 112, Issue 8, 3444-3454, October 15, 2008
The tetraspanin CD63 is involved in granule targeting of neutrophil elastase
Blood Källquist et al.
112: 3444
Supplemental materials for: Kallquist et al
Files in this Data Supplement:
- Figure S1. ProNE and GFP do not form complexes (JPG, 57.5 KB)
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COS cells were cotransfected with plasmids carrying GFP (GFP), GFP CD63 (CD63), or NE (NE), and cell lysates were examined by IP/Western. This was performed to show that the GFP part of the GFP-CD63 fusion protein did not by itself bind proNE. In support of this, coprecipitation is shown between CD63 and proNE but not between GFP and proNE (row 1). Anti-GFP detected both GFP and GFP-CD63 (row 2). In addition, negative control IP with non-immune serum is shown (rows 34).
- Figure S2. NE mRNA shows inter-clonal variation in CD63-depleted HL-60 cells (JPG, 21.1 KB)
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Relative mRNA levels measured by real-time PCR with 18S mRNA as an internal control are compared with wild-type (wt) cells and 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 ± SD of 1 representative experiment out of 3 similar experiments, each done in triplicate from 1 run of real-time PCR, and calculated according to the ΔΔCT method.
- Figure S3. Scrambled siRNA control (JPG, 174 KB)
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(A) The CD63 mRNA expression is not affected by scrambled siRNA in HL-60 cells real-time PCR. Relative CD63 mRNA levels are compared to wild-type (wt) cells and scrambled siRNA (siRNA control 26), CD63 siRNA mock (clone B8), and CD63 siRNA (clone C14). Data presented are mean ± SD of 1 representative experiment of 3 similar experiments done each in triplicate from 1 run of real-time PCR, and calculated according to the ΔΔCT method. The mean (SD range) CD63 mRNA expression of the 5 siRNA control clones was 1.1 (0.921.25), not significantly different from the wild-type cells. (B) The NE mRNA expression shows inter-clonal variation in HL-60 cells transfected with scrambled siRNA real-time PCR. Relative NE mRNA levels are compared with wild-type cells, as in (A). The mean (SD range) NE mRNA expression of the 5 scrambled siRNA control clones was 0.7 (0.480.73). (C) The proNE protein level shows a low inter-clonal variation in HL-60 cells transfected with scrambled siRNA Western blotting. Immunoblotting of cell lysates was performed with anti-proNE and anti-actin. Quantitative measurement of proNE protein was performed by densitometry. The numbers represent the relative values, normalized for wild-type (wt) cells. The proNE of scrambled siRNA control clones varied less than the corresponding NE mRNA. Densitometry showed a mean of 0.7 for the siRNA clones compared to wild-type cells. The molecular weights (kDa) for each SDS-PAGE are shown to the left. (D) CD63 and NE were not affected by scrambled siRNA in HL-60 cells immunofluorescence microscopy. Scrambled siRNA control clone 2 was compared with wild-type (wt) cells. Cells were stained with antibodies against CD63 or NE (detecting both proNE and mature NE), and examined. siRNA control cells showed no change in the expression of CD63, compared to wild-type cells. The staining of NE was weaker than for CD63, but no significant difference between siRNA control and wild-type cells could be observed. Identical staining protocols and microscope and camera settings were used when comparing the clones. (E) CD63 is not affected by scrambled siRNA in HL-60 cells but NE is slightly lower than in wild-type cells flow cytometry. Scrambled siRNA control clone 2 was compared with wild type (wt) cells. The histograms show flow cytometry analysis of both non-permeabilized and permeabilized cells stained for CD63 and NE in siRNA control clone 2. The mean fluorescence and geometric mean of 5 siRNA control clones were compared, and control clone 2 was selected as representative. The variation between clones was low, in regards to both NE and CD63 expression. (F) ProNE processing and constitutive secretion is unaffected by scrambled siRNA in HL-60 cells biosynthetic radiolabeling. Scrambled siRNA control clone 5 was compared to wild-type (wt) cells. Transfected HL-60 cells were radiolabeled (pulse) for 30 minutes, followed by chase of the radiolabel for up to 4 hours. At the indicated time points, 20 × 106 cells and incubation medium were withdrawn, extracted, immunoprecipitated, and examined as described in Materials and methods. The numbers to the left are molecular mass standards. (G) Granule morphology is unaffected by scrambled siRNA in HL-60 cells immunoelectron microscopy. Ultrathin cryosections of cells transfected with scrambled siRNA were labeled with rabbit anti-MPO (10-nm gold) or with rabbit anti-NE (10-nm gold), as indicated. The cells show electron-dense granules labeled for MPO and NE.
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