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Blood, Vol. 111, Issue 4, 2290-2299, February 15, 2008
Semaphorin3A signaling controls Fas (CD95)-mediated apoptosis by promoting Fas translocation into lipid rafts
Blood Moretti et al.
111: 2290
Supplemental materials for: Moretti et al
Files in this Data Supplement:
- Figure S1. Pro-apoptotic effect of Sema3A is independent from Rap1 and protein synthesis (JPG, 92.1 KB)
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(A) Cell surface expression of Fas was determined by immunofluorescence flow cytometry in cells treated with control IgG or Sema3A-Fc for 6 hrs. (B) Jurkat cells were preincubated for 20 min in the absence or in presence of 100 ng/ml cycloheximide (CHX) or 15 ng/ml actinomycin D (Act. D), and then treated with CH11 mAb alone or plus Sema3A. Cell death was analyzed by flow cytometry with propidium iodide. (C) Jurkat cells were transfected with an empty vector (pcDNA3.1) or a vector containing cDNA of a dominant-negative Rap1 (Rap1N17). Then, stable transfectants were stimulated with 200 ng/ml cytotoxic anti-Fas CH11 mAb in the absence or presence of Sema3A-Fc (150 ng/ml) for 6-8 hrs. Cell death was determined by flow cytometry with annexin V staining.
- Figure S2. The presence of Sema3A controls Fas-induced cell death in primary cells (JPG, 105 KB)
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(A) Bone marrow leukemic primary cultures derived from patients with M2 or M3 leukemia were incubated with rhFasL, rhFasL plus Sema3A-Fc, MBCD (15 µg/ml), or rhFasL plus Sema3A-Fc in the presence of 10 mM MBCD for 3 hrs. Cell death was quantified by flow cytometry with annexin V staining. (B) Alternatively, cells were then incubated for 30 min with FAM-VAD-fmk and caspase activation was analyzed by flow cytometry. Results represent the means and errors of three independently repeated experiments. Statistical significance is shown.
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