Figure 2

Identification of HMGB1 releasing agents from chemical libraries. (A) U2OS cells stably co-expressing streptavidin-NLS3 and HMGB1-SBP-GFP were seeded in 384-well plates either in the absence or in the presence of biotin before treatment with 1200 small molecules from the Prestwick chemical library (most of which are approved by FDA, EMA and other agencies) at a final concentration of 10 µM for 48 h. Following HMGB1-GFP fluorescence was quantified in the nucleus as well as the cytoplasm and, based on Hoechst 33342 staining, nuclear pyknosis was assessed as an indicator for cell death. Quantitative data were normalized by z-scoring (mean, n = 4) hierarchically clustered and depicted as heat map, with red and blue values indicating positive and negative effects, respectively. The top 20 compounds are shown. (B) U2OS cells stably co-expressing streptavidin-NLS3 and HMGB1-SBP-GFP were pre-incubated or not with biotin before treatment with a collection of FDA-approved anticancer agents at different concentrations for 48 h. Following cytoplasmic HMGB1-SBP-GFP fluorescence was quantified. Obtained results were hierarchically clustered (average linkage*, similarity metric using pearson distance*) and depicted as a heat map. Agents from the custom library have been used at the following concentrations azacitidine (1, 5, 10, 20 µM); paclitaxel (0.1, 0.5, 1, 2 µM); docetaxel, (0.1, 0.5, n 1, 2 µM); oxaliplatin (10, 50, 100, 200 µM); decitabine (1, 5, 10, 20 µM) and suberoylanilide hydroxamic acid (SAHA; 1, 5, 10, 20 µM).