Figure 2.

High throughput screening of the Prestwick chemical library. Human osteosarcoma U2OS cells coexpressing streptavidin-KDEL and ss-SBP-GFP were seeded in 384-well plates and treated by the compounds of the Prestwick library at 10 and 20 μM for 4 h. Without removing the drugs, biotin was added at 40 μM and incubated for another 4 h before cells were fixed for image acquisition (A). Brefeldin A (BFA) was used as positive control for conventional secretion inhibition. Representative images of cells without GFP release in the absence of biotin, with GFP release in the presence of biotin, as well as with inhibited GFP release by pretreatment of BFA and in the presence of biotin, are depicted in (B); scale bar equals 10 μm. Average cytoplasmic GFP intensity was quantified and data was normalized by Z-scoring. Data from two independent experiments were summed up to obtain a cumulative score for each agent (C) or were hierarchically clustered and depicted as a heat map in which each block represents the average value obtained for each treatment (mean, n = 4) (D). Compounds belonging to the top 2.5% percentile of the resulting ranking are shown.