Fig. 8

Sequential two-step release of HMGB1 from cells undergoing necroptosis. a, b L929 cells stably expressing HMGB1-mCherry or histone H3-mCherry were stimulated with TZ. The signals of HMGB1- or histone H3-mCherry were analyzed every 2 min. Representative images of HMGB1-mCherry (a) or histone H3-mCherry (b) (upper), SYTOX Green (middle), and merged images of DIC and Hoechst (lower) (left) (n = 5 cells). Scale bars, 20 μm. Intensities of mCherry (magenta) and SYTOX Green (green) were quantified and relative intensities were plotted at the indicated times (right). c, d L929-SMART cells were transiently transfected with HMGB1-mCherry and stimulated with TZ. The FRET/CFP ratio was analyzed as in Fig. 1b. Representative images of the FRET/CFP ratio (upper), HMGB1-mCherry (middle), and merged images of DIC and HMGB1-mCherry (lower) (n = 5 cells) (c). White arrowheads indicate released HMGB1-mCherry in the cytoplasm. Scale bars, 20 μm. ΔFRET/CFP ratio (left), and relative intensity of HMGB1-mCherry in the nucleus and the cytoplasm (right) were plotted at the indicated times after stimulation (d). Intensities of HMGB1-mCherry in the cytoplasm were determined by subtracting nuclear intensities of HMGB1-mCherry from intracellular (total) intensities of HMGB1-mCherry. Two vertical dotted lines indicate the times when nuclear efflux of HMGB1-mCherry started and extracellular release of HMGB1-mCherry was terminated, respectively. All results are representative of at least three independent experiments