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Blood, Vol. 112, Issue 13, 4961-4970, December 15, 2008
CaMKII promotes TLR-triggered proinflammatory cytokine and type I interferon production by directly binding and activating TAK1 and IRF3 in macrophages
Blood Liu et al.
112: 4961
Supplemental materials for: Liu et al
Files in this Data Supplement:
- Figure S1. TLR3, 9 ligands induce intracellular Ca2+ release in macrophages (JPG, 136 KB)
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RAW264.7 cells were loaded with Fluo 3/AM and stimulated with 0.3 µM CpG ODN (A) or 10 µg/ml poly(I:C) (B) respectively in the absence or presence of 1.5 mM EGTA or 6 µM BAPTA-AM, and then imaged by confocal microscopy at 10-s intervals. The basal (left panel) and peak (middle panel) fluorescence are displayed. Graphs (right panel) showed changes in mean fluorescence intensity from 15 cells per microscope field over time. Data are representative of three independent experiments. Original magnification of images: ×100.
- Figure S2. TLR ligands induce CaMKII activation in macrophages (JPG, 68.1 KB)
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RAW264.7 cells were stimulated with LPS (0.1 µg/ml), CpG ODN (0.3 µM) or Poly(I:C) (10 µg/ml) respectively for the indicated time. Total expression (A) or phosphorylation (T286) (B) of CaMKIIα was detected by Western blot. Similar results were obtained in three independent experiments.
- Figure S3. Blockade of CaMKII activation with KN62 attenuates TLR4-, TLR9-, and TLR3-activated proinflammatory cytokine and IFN-β production in macrophages (JPG, 92.4 KB)
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(A) RAW264.7 cells (4 × 105) were pretreated with the indicated different doses of KN62 for 30 min followed by stimulation with 0.1 µg/ml LPS for 8h. The production of IL-6 was measured by ELISA. (B) RAW264.7 cells (4 × 105) were treated with 15 µM KN62 for the indicated time, and cell proliferation were measured with MTT assays. (C–E) RAW264.7 cells (4 × 105) were pretreated with 15 µM KN62 for 30 min followed by stimulation with 0.1 µg/ml LPS (C), 0.3 µM CpG ODN (D) or 10 µg/ml Poly(I:C) (E) for the indicated time. The production of IL-6, TNF-α or IFN-β was measured by ELISA. Data are shown as mean ± SD of three independent experiments. *, P < .05; **, P < .01.
- Figure S4. Silencing of CaMKII expression attenuates TLR4-, TLR9-, and TLR3-activated proinflammatory cytokine and IFN-β production in macrophages (JPG, 89.2 KB)
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RAW264.7 cells (1.5 × 105) were transfected with control small RNA (Ctrl), CaMKIIα siRNA1 (A, D, E) or siRNA2 (C). After 48 hr, the cells were stimulated with 0.1 µg/ml LPS (A, C), 0.3 µM CpG ODN (D) or 10 µg/ml Poly(I:C) (E) for the indicated time. IL-6, TNF-α, or IFN-β in the supernatants was measured by ELISA. (B) RAW264.7 cells were transfected with control small RNA (Ctrl) or CaMKIIα siRNA2. After 48 hr, CaMKIIα and β-actin expression in the cells was detected by immunoblot. Data are shown as mean ± SD of three independent experiments. *, P < .05; **, P < .01.
- Figure S5. Activation of CaMKII enhances IRF3 activation in TLR3-triggered macrophages (JPG, 50.9 KB)
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RAW264.7 cells were transfected with 100 ng IRF3 luciferase reporter plasmids (80 ng Gal4 luciferase reporter plasmid, 20 ng Gal4-IRF3 expressing plasmid), 10 ng of pTK–Renilla-luciferase, together with indicated amount of CaMKII290 plasmid. Total amounts of plasmid DNA were equalized using empty control vector. After 36 h of culture, the cells were stimulated with 10 µg/ml Poly(I:C) for 6 h. Luciferase activity was measured and normalized by Renilla luciferase activity. Data are shown as mean ± SD (n = 5) of one typical experiment. Similar results were obtained in three independent experiments.*, P < .05; **, P < .01.
- Figure S6. CaMKII doesn’t bind IRAK1 (JPG, 50.8 KB)
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RAW264.7 cells were stimulated with 0.1 µg/ml LPS for the indicated time. Equal amount cell lysates were immunoprecipitated (IP) with CaMKIIα antibody and then immunoblotted (IB) with IRAK1 and CaMKIIα antibody.
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