Figure 3.

Compound testing in orthogonal and membrane trafficking assays. A, β-hexosaminidase secretion from RBL-2H3 cells in 15 min was determined for untreated (control) or ionomycin (iono)-treated cells without or with the indicated compound at 20 μm provided 15 min prior to ionomycin stimulation. Control values were subtracted and results expressed as percent β-hexosaminidase secretion as mean ± S.D. (n = 3). B, percent β-hexosaminidase secretion in 15 min from control or ionomycin-stimulated bone marrow–derived mast cells treated with the indicated concentrations of bexin-1 for 15 min are shown as mean ± S.D. (n = 3). C, RBL-2H3 cells expressing TNFα-pHluorin were left untreated or stimulated for 15 min with ionomycin. Cells were stained with Hoechst 33342, and cell surface fluorescence was quantitated by epifluorescence microscopy. Values for cells treated for 15 min with the indicated compound (20 μm) are shown as percent maximal fluorescence (mean ± S.D., n = 3). D, endocytosis was determined by the uptake of transferrin–Texas Red conjugate (Tf-Red). Cells were treated with 20 μm compounds for 15 min, followed by 15-min incubation with Tf-Red. Cells were washed, fixed, and imaged by confocal microscopy. Shown is a representative study of triplicate determinations (*, p < 0.05; **, p < 0.01). E, Alamar Blue was used to monitor cell viability upon exposure of live cells to inhibitors at the indicated concentrations for 5 h. The various compounds tested correspond to classes A (F5128-0085, bexin-1 and F5085-0061, bexin-5), B (F2927-0504), and D (F4448-0530) as representative of many compounds tested. For class F, we tested 13 compounds, with the most inhibitory members from the cell screen (F5024-0157 and F2590-0733) shown here.