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Blood, Vol. 110, Issue 6, 1942-1949, September 15, 2007
Small-molecule agonists of SHIP1 inhibit the phosphoinositide 3-kinase pathway in hematopoietic cells
Blood Ong et al.
110: 1942
Supplemental materials for Ong et al, Vol. 110, Issue 6, 1942-1949
Files in this Data Supplement:
- Document 1. Supplemental methods (19.9 KB)
- Figure S1. SHIP1 activating activity of various marine invertebrate extracts (PDF, 37.3 KB) -
Desiccated marine invertebrate methanolic extracts were resuspended at approximately 1 mg/mL in DMSO and tested at 40 µg/mL in the in vitro SHIP1 enzyme assay as described in “Materials and methods, In vitro SHIP enzyme assay.” Approximately 10 extracts showed SHIP1 activating activity out of 2000 extracts screened.
- Figure S2. LY294002 inhibits TNF
 expression in both SHIP1+/+ and SHIP1−/− macrophages (PDF, 29 KB) -
SHIP1+/+ (▒) and SHIP1−/− (▓) bone marrow derived macrophages were pretreated with LY294002 or carrier for 30 min prior to stimulation with 10 ng/mL of LPS at 37°C; TNF levels were then determined by ELISA. Absolute TNF levels for SHIP1+/+ and SHIP1−/− cells were 693 ± 37 pg/mL and 921 ± 21 pg/mL, respectively.
- Figure S3. LY294002 inhibits calcium flux in both SHIP1+/+ and SHIP1−/− mast cells (PDF, 59 KB) -
SHIP1+/+ and SHIP1−/− bone marrow derived mast cells, pretreated overnight with 0.1 g/ml IgE (SPE-7), were incubated for 30 min with the indicated concentration of LY294002. Cells were then stimulated or not (■■■■) with 5 ng/mL DNP-HSA (arrow) and intracellular calcium levels monitored over time by spectrofluorometry.
- Figure S4. LY294002 but not AQX-016A inhibits PKB phosphorylation in LnCAP cells (PDF, 18.1 KB) -
The nonhemopoietic, prostate cancer LnCAP cell line was treated with AQX-016A or LY294002 for 30 minutes and cell lysates analyzed for phospho-PKB-Thr 308 or phospho-PKB 473 as described in “Materials and methods, LPS stimulation of macrophages.”
- Figure S5. AQX-016A inhibits DNFB induced inflammation in a mouse ear edema/cutaneous anaphylaxis model (PDF, 13.3 KB) -
CD1 mice were sensitized to the hapten DNP by cutaneous application of 25 µL of 0.5% (DNFB) in acetone to the shaved abdomen of mice for two consecutive days. Tritiated thymidine (3H-Tdr, 20 µCi, GE Healthcare) was injected intraperitonially one week after the first DNFB application. 3H-Tdr labels rapidly dividing cells of the mouse, including neutrophils.1 Twenty-four hours later, test substances (dissolved in 10 µL of 1:2 DMSO:MeOH) were painted on the right ear while the left ear received vehicle control. Thirty minutes after drug application, DNFB was applied to both ears to induce mast cell degranulation. The resulting inflammatory cell infiltration was quantified by taking a 6mm diameter punch from the ear 1 hour later for dissolution in Solvable (Perkin Elmer-Packard, Woodbridge, ON) and liquid scintillation counting as described.1 The ability of test substances to inhibit mast cell degranulation was then determined by calculating the ratio of 3H-Tdr in the test (right) ear vs the control (left) ear as described.1 One group of mice had DNFB applied only to the left ear, leaving the right ear noninflamed, in order to control for basal 3H-Tdr incorporation into ear parenchymal cells. (In later AQX-MN100 experiments Fig 4E, ear inflammation was measured by quantifying neutrophil specific myeloperoxidase.)
- Figure S6. Wild-type but not C2 domain-deleted SHIP1 enzyme binds AQX-MN100 (PDF, 20.8 KB) -
Copper chelate (His-Tag) YSi SPA Scintillation Beads coated with either wild-type (WT) or C2 domain deleted (ΔC2) SHIP1 enzyme in the presence of 0.25% BSA as described in “Materials and methods, Scintillation proximity assays“ were aliquoted into 96 well plates. Five µCi of 3H-AQX-MN100 (42 Ci/mmol) was then added and the mixture incubated with shaking at 23°C in the dark. The amount of 3H-AQX-MN100 interacting with the protein coated beads was quantified on a plate scintillation counter.
- Figure S7. AQX-MN100 has minimal activities on a panel of protein kinase and phosphatases (PDF, 62.4 KB) -
Compound profiling activity was undertaken using 100 protein kinase and phosphatase targets by SignalChem (Richmond, BC) against compound AQX-MN100 (2 µM final concentration). Protein kinase assays were performed in the presence of 50 µM ATP at 30°C for 15 minutes. Protein phosphatase activites were determined using pNPP as substrate and were also performed at 37°C for 15 minutes. The activity of the enzymes in the presence of AQX-MN100 was compared to that in the vehicle control and expressed (A) as a percentage change in activity relative to that observed in the vehicle control. Changes in activity of less than 25% were not considered significant. Enzymes affected by AQX-MN100 are plotted in an expanded graph in panel B. This experiment was funded by Aquinox Pharmaceuticals.
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