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. Author manuscript; available in PMC: 2014 Jun 27.

Published in final edited form as: J Med Chem. 2013 Jun 6;56(12):4990–5008. doi: 10.1021/jm400248c

Effects of compound 8b in mast cells. (A) Compound 8b decreases NF-AT luciferase activity in BMMC. 8×10⁶ cells PEP^+/+ and PEP^−/− BMMC were transfected with 4.5 µg 3xNF-AT-luciferase and 0.8 µg of Renilla luciferase expression. 24 h after transfection, the cells were sensitized with anti-DNP IgE for 18 h. Cells were treated with C28 (5 µM) or 8b (5 µM, 20 µM) for 1 h to the end of IgE incubation. Cells were then activated with DNP-HSA (200 ng/ml) for 8 h and firefly luciferase activity was measured and normalized to Renilla luciferase activity. Results are presented as the mean ± SEM (n=3). PEP^+/+ ; PEP^−/− . Results in bar charts are presented as the mean ± SEM (***p≤0.0001, n=3) (B). Compound 8b reduces mast cell degranulation in BMMC. 5×10⁶ cells/ml BMMC from PEP^+/+ and PEP^−/− mice were sensitized with anti-DNP-IgE for 16 h and treated with C28 (5 µM), 8b (5 µM) or 8b (20 µM) for 1 h at the end of IgE incubation. These cells were then activated with DNP-HSA(200 ng/ml) for various time points. BMMC were pelleted and the amount of β-hexosaminidase in the supernatant and in the solubilized pellet were measured using p-nitrophenyl N-acetyl-β-D-glucosaminidine (p-NAG) as a substrate. Percent degranulation was calculated as follows: (released activity/total activity) ×100. The results are represented as the mean of ± SEM of 3 independent experiments (***p≤0.0005, n=3). PEP^+/+ ; PEP^−/− ; C28 (5µM) ; 8b (5µM) ; 8b (20µM) . The statistics were calculated using unpaired, 2-tailed, Student’s t-test.

Inline graphic — Effects of compound 8b in mast cells. (A) Compound 8b decreases NF-AT luciferase activity in BMMC. 8×10⁶ cells PEP^+/+ and PEP^−/− BMMC were transfected with 4.5 µg 3xNF-AT-luciferase and 0.8 µg of Renilla luciferase expression. 24 h after transfection, the cells were sensitized with anti-DNP IgE for 18 h. Cells were treated with C28 (5 µM) or 8b (5 µM, 20 µM) for 1 h to the end of IgE incubation. Cells were then activated with DNP-HSA (200 ng/ml) for 8 h and firefly luciferase activity was measured and normalized to Renilla luciferase activity. Results are presented as the mean ± SEM (n=3). PEP^+/+ ; PEP^−/− . Results in bar charts are presented as the mean ± SEM (***p≤0.0001, n=3) (B). Compound 8b reduces mast cell degranulation in BMMC. 5×10⁶ cells/ml BMMC from PEP^+/+ and PEP^−/− mice were sensitized with anti-DNP-IgE for 16 h and treated with C28 (5 µM), 8b (5 µM) or 8b (20 µM) for 1 h at the end of IgE incubation. These cells were then activated with DNP-HSA(200 ng/ml) for various time points. BMMC were pelleted and the amount of β-hexosaminidase in the supernatant and in the solubilized pellet were measured using p-nitrophenyl N-acetyl-β-D-glucosaminidine (p-NAG) as a substrate. Percent degranulation was calculated as follows: (released activity/total activity) ×100. The results are represented as the mean of ± SEM of 3 independent experiments (***p≤0.0005, n=3). PEP^+/+ ; PEP^−/− ; C28 (5µM) ; 8b (5µM) ; 8b (20µM) . The statistics were calculated using unpaired, 2-tailed, Student’s t-test.