Figure 1

An optimized fluorescent biosensor–based cell line for the identification of HMGB1 releasing agents. (A) Scheme of the RUSH (Retention Using Selective Hooks)-HMGB1 cell assay. U2OS cells stably expressing Streptavidin-NLS3 (Str-NLS3) as a hook that localizes in the nuclear dots and can be detected with an anti-streptavidin antibody ((B) red color). In the absence of biotin, HMGB1-SBP-GFP is retained in the nucleus due to the streptavidin-SBP interaction and co-localizes with Str-NLS3. Upon biotin addition, the HMGB1-SBP-GFP reporter is released from the hook and diffuses in the nucleus, while the hook remains punctiform. Granularity of HMGB1-GFP and streptavidin-AF568 was quantified after the cells were exposed to biotin (C). (D) HMGB1 releasing agent mitoxantrone (MTX, 2 µM) induced the exodus of HMGB1 only in the presence of biotin. Cytoplasmic HMGB1 was quantified after the cells were exposed to MTX either in the absence or presence of biotin (E). Data are reported as means ± SEM at 24 h post treatment (n = 4; *P < 0.05, **P < 0.01, and ***P < 0.001, two-tailed Student’s t test, compared to control cells). Scale bar = 10 µm.