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Blood, Vol. 111, Issue 1, 243-250, January 1, 2008
Interaction between Hck and HIV-1 Nef negatively regulates cell surface expression of M-CSF receptor
Blood Hiyoshi et al.
111: 243
Supplemental materials for: Hiyoshi et al
Files in this Data Supplement:
- Figure S1. Nef inhibits M-CSF-dependent growth of TF-1-fms-Nef-ER cells but not GM-CSF-dependent growth of TF-1-Nef-ER cells (JPG, 69.1 KB)
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(A) The growth of TF-1, TF-1-Nef-ER (clone #5), TF-1-fms, or TF-1-fms-Nef-ER cells (clone #9) was analyzed by the MTT assay with varying concentrations of 4-HT (0, 0.01, and 0.1 µM). TF-1 and TF-1-Nef-ER cells seeded at a density of 2 × 104 cells/mL were cultured in the presence of 2 ng/mL GM-CSF for 4 days. TF-1-fms and TF-1-fms-Nef-ER cells seeded at a density of 1 × 104 cells/mL were cultured in the presence of 100 ng/mL M-CSF for 3 days. Differences between untreated TF-1-Nef-ER cells and those treated with 0.1 µM 4-HT were significant (p = 0.001, Student’s t-test). Results are representative of three independent experiments with similar results. Data shown are the mean ± SD from triplicate assays. TF-1, TF-1-Nef-ER (clone #5), TF-1-fms, or TF-1-fms-Nef-ER cells (clone #9) were analyzed for the expression of Nef-ER by Western blotting. The total cell lysates used were obtained from an equal number of cells. (B) The growth of TF-1 and TF-1-Nef-ER cells (clones #5 and #15) was analyzed by the MTT assay with varying concentrations of GM-CSF (0, 0.3, 1.0 and 3.0 ng/mL). These cells were seeded at a density of 1 × 104 cells/mL and cultured in the absence or presence of 0.1 µM 4-HT for 4 days. The level of Nef-ER in these cells is shown. (C) The growth of TF-1-fms or TF-1-fms-Nef-ER cells (clones #3, #8, and #9) was analyzed by the MTT assay with varying concentrations of 4-HT (0, 0.01, and 0.1 µM). These cells seeded at a density of 1 × 104 cells/mL were cultured in the presence of 100 ng/mL M-CSF for 3 days. The level of Nef-ER in these cells is shown. (A and B) Results are representative of three independent experiments with similar results. Data shown are the mean ± SD from triplicate assays.
- Figure S2. M-CSF but not GM-CSF induces the down-regulation of Fms (JPG, 23.4 KB)
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(A) TF-1-fms-Nef-ER cells were pre-cultured with M-CSF-free media for 12 hours and then stimulated with M-CSF at the indicated concentrations for 2 hours. The surface expression of Fms was analyzed by flow cytometry with PE-labeled anti-Fms antibody. The level of Fms was expressed as the mean fluorescence intensity (MFI). Data shown are the mean ± SD from triplicate assays. (B and C) Macrophages prepared by culturing monocytes with 100 ng/mL M-CSF for 5 days were pre-cultured with M-CSF-free media for 4 hours and then stimulated with M-CSF at the indicated concentrations for 2 hours. Alternatively, macrophages pre-cultured with M-CSF-free media were left untreated, or stimulated with M-CSF (100 ng/mL) or GM-CSF at the indicated concentrations for 2 hours. The surface expression of Fms was analyzed by flow cytometry with PE-labeled anti-Fms antibody. The level of Fms was expressed as the MFI. Data shown are the mean ± SD from duplicate assays.
- Figure S3. Expression of CD14 and Fms in macrophages used for the nucleofection experiments (JPG, 39.9 KB)
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Macrophages prepared by culturing monocytes with 100 ng/mL M-CSF for 5 days were analyzed for their expression of CD14 and Fms. PE-labeled anti-CD14 purchased from PharMingen was used for the flow cytomteric analysis.
- Figure S4. gp130Fms appearing in 293 cells co-expressing Nef/Hck is Golgi-localized Fms (JPG, 52.3 KB)
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The transfection and immunofluorescence analyses were performed as in Figure 4A, by using another 293-Fms clone. The transfected cells were co-stained with anti-Fms antibody (green), anti-GM130 antibody (red), and DAPI (blue). Anti-Vti1a antibody purchased from BD Biosciences was also used.
- Figure S5. Interaction between Nef and Hck (JPG, 26.6 KB)
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An SF2-derived Nef gene was cloned into pGEX-6P-2 vector (GE Healthcare), and GST-Nef fusion proteins or control GST proteins were expressed in BL21 (GE Healthcare). Then, the cleared bacterial lysates were incubated with GST-Bind Resin (Novagen). In the left panel, GST or GST-Nef proteins bound to the resin were analyzed by SDS-PAGE followed by Coomassie brilliant blue (CBB) staining. In the right panel, a GST pull-down analysis was performed upon incubation of GST- or GST-Nef-bound resin with total cell lysate of 293 cells transfected with the Hck expression plasmid. After extensive washing, Hck bound to the resin was analyzed by Western blotting (W.B.). Two percent of the total cell lysate was analyzed in parallel (Input).
- Figure S6. Co-localization of Nef and active Hck in the perinuclear compartment of macrophages nucleofected with CD8-Nef (JPG, 24.2 KB)
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Macrophages were nucleofected with CD8-Nef plasmid and co-stained with anti-Hck pTyr411 antibody (green), anti-Nef antibody (red), and DAPI (blue), as described in Figure 6. The anti-Nef mouse monoclonal antibody (clone EH1) obtained from the National Institute of Health AIDS Research and Reference Reagent Program was used for the co-staining.
- Figure S7. Nef down-regulates a Fms mutant lacking the entire intracellular region in the presence of Hck (JPG, 29.8 KB)
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A mutant Fms cDNA with a complete deletion of the intracellular region was prepared by introducing the stop codon at 538Y. TM indicates the transmembrane region of Fms. Parental 293 cells were transfected with the Fms plasmid, alone or in combination with the plasmid for Nef or Hck. Total cell lysate was prepared and subjected to Western blotting with antibodies to Fms, Nef, Hck or ERK.
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