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Blood, Vol. 113, Issue 13, 2945-2954, March 26, 2009
SHIP prevents lipopolysaccharide from triggering an antiviral response in mice
Blood Sly et al.
113: 2945
Supplemental materials for: Sly et al
Files in this Data Supplement:
- Figure S1. Cells treated ± SHIP or non-silencing siRNA (JPG, 63.5 KB)
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(A) RAW246.7 cells were treated ± SHIP or non-silencing siRNA for 24 hr and the cells were stimulated with 10 ng/ml LPS for 24 hr. Whole cell lysates were then subjected to immunoblot analyses for SHIP and GAPDH. (B) RAW246.7 cells were treated ± SHIP or non-silencing siRNA for 24 hr, then pre-treated or not with 10 ng/ml LPS or 30 nM CpG for 24 hr and challenged with 10 ng/ml LPS or 30 nM CpG for 24 hr. Cell supernatants were assessed for IL-6 and TNFα. Results are the mean ± SEM for 3 independent experiments assayed in duplicate.
- Figure S2. RAW246.7 cells were treated ± SHIP or non-silencing siRNA for 24 hr, then pre-treated or not with 10 ng/ml LPS for 24 hr and challenged with 5 µg/ml dsRNA (left panel) or 10 ng/ml LPS (right panel) for 24 hr (JPG, 40.2 KB)
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Cell supernatants were assessed for IFNβ levels. Results are the mean ± SEM for 3 independent experiments assayed in duplicate. It is worthy of note that SHIP siRNA treatment did not increase IFNβ secretion following dsRNA pretreatment, consistent with the siRNA treatment not lowering SHIP levels below its level in unstimulated cells.
- Figure S3 (JPG, 71.6 KB)
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(A) Wild type BMmφs were not pre-treated (C), pre-treated with 0.1% DMSO (sol) or pre-treated with 14 µM LY or 50 nM W and 3 additional serial 4-fold dilutions of these inhibitors for 30 min prior to stimulation with 30 nM CpG (left hand panels) or 10 ng/ml LPS (right hand panels) and 24 hr cell supernatants assessed for TNFα (upper panels) and IL-6 (lower panels) by ELISA. (B) Wild type BMmφs were not pre-treated (C), pre-treated with 0.1% DMSO (sol) or pre-treated with 10 µM or 3 additional serial 4-fold dilutions of the PI3K p110 isoform specific inhibitor indicated for 30 min prior to stimulation with 30 nM CpG (left hand panels) or 10 ng/ml LPS (right hand panels) and 24 hr cell supernatants assessed for TNFα (upper panels) and IL-6 (lower panels) by ELISA. Results shown are the mean ± SEM of 3 independent experiments assayed in duplicate.
- Figure S4. Structures of the p110 isoform specific PI3K inhibitors (JPG, 110 KB)
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- Figure S5 (JPG, 74.4 KB)
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(A) Wild type BMmφs were not pre-treated (C), pre-treated with 0.1% DMSO (sol) or pre-treated with 14 µM LY or 50 nM W and 3 additional serial 4-fold dilutions of these inhibitors for 30 min prior to stimulation with 30 nM CpG (left hand panel) or 10 ng/ml LPS (right hand panel) and 24 hr cell supernatants assessed for IFNβ by ELISA. (B) Wild type BMmφs were not pre-treated (C), pre-treated with 0.1% DMSO (s or sol) or pre-treated with 10 µM or 3 additional serial 4-fold dilutions of the PI3K p110 isoform specific inhibitor for 30 min prior to stimulation with 5 µg/ml dsRNA (top graph) or 10 ng/ml LPS (bottom graph) and 24 hr cell supernatants assessed for IFNβ by ELISA. PI3K isoform specific inhibitors numbered 1-7 were: 1, PIK-90; 2, PIK-93; 3, PI-103; 4, TGX-221; 5, SW18; 6, SW30; 7 AS605240. Results shown are the mean ± SEM of 3 independent experiments assayed in duplicate.
- Figure S6 (JPG, 88.5 KB)
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(A) SHIP+∕+ (white bars) and −∕− (black bars) Pmφs were treated or not with 14 uM LY or 50 nM W for 30 min and then with 30 nM CpG (left panels) or 10 ng/ml LPS (right panels) for 24 hr. Cell supernatants were assessed for TNFα (top panels) or IL-6 (bottom panels). (B) SHIP+∕+ (white bars) and −∕− (black bars) Pmφs were pre-treated with 10 µM PI3K p110 isoform specific inhibitors 1 through 7 or with 0.1% DMSO (sol) or without (C) vehicle control for 30 min prior to stimulation with 30 nM CpG (left panels) or 10 ng/ml LPS (right panels) and 24 hr cell supernatants assessed for TNFα (top panels) and IL-6 (bottom panels) by ELISA. Results are the means ± SEM of 3 independent experiments assayed in duplicate. *p<0.05.
- Figure S7 (JPG, 30 KB)
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(A) SHIP+∕+ (white bars) and −∕− (black bars) BMmφs were untreated (0) or treated with 30 nM CpG, 5 µg/ml dsRNA or 10 ng/ml LPS and RNA isolated 24 hr later to estimate TLR3 mRNA levels using Q-PCR. Relative gene expression was normalized to unstimulated cells. Results shown are the mean ± SEM of 4 independent experiments assayed in duplicate. *p<0.03 comparing stimulated to unstimulated cells; **p<0.01 comparing stimulated to unstimulated cells and comparing SHIP−∕− to SHIP+∕+ cells; stimulation with dsRNA was not significantly different from stimulation with LPS and stimulation with CpG was not significantly different from unstimulated in either the SHIP+∕+ or SHIP−∕− cells. (B) SHIP+∕+ (white bars) and −∕− (black bars) BMmφs were untreated (0) or treated with 30 nM CpG, 5 µg/ml dsRNA or 10 ng/ml LPS for 24 hr and then stimulated with 30 nM CpG for 24 hr. Cell supernatants were harvested and assessed for IFNβ by ELISA. Unstimulated cells did not produce detectable IFNβ. Results shown are the mean ± SEM of 3 independent experiments assayed in duplicate. *p<0.05 compared to untolerized cells, **p<0.02 compared to untolerized cells and compared to SHIP+∕+ cells. NS = not significantly different from untolerized cells.
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