Fig. 9

LCI-S uncovers two different modes of the release of HMGB1. a–c L929-SMART cells were transiently transfected with HMGB1-mCherry and then stimulated with TZ. The FRET/CFP ratio was analyzed as in Fig. 1b. Representative images of the FRET/CFP ratio (Ratio), intracellular (Epi), and extracellularly released (TIRF) HMGB1-mCherry (a) (n = 19 cells). Epi shows merged images of bright field and intracellular HMGB1-mCherry. Scale bar, 25 μm. Representative temporal relationship between MLKL activation and extracellular release of HMGB1-mCherry (b). The FRET/CFP ratio and the signals of extracellularly released HMBG1-mCherry were plotted at the indicated times after stimulation. Two vertical dotted lines at 94 and 142 min indicate initiation of an increase in the FRET/CFP ratio and extracellular release of HMGB1-mCherry, respectively. Average intervals between the initiation of the increase in the FRET/CFP ratio and extracellular release of HMGB1 (c) (n = 19 cells). d–g Two different modes of extracellular release of HMGB1 during necroptosis. L929-HMGB1-mCherry cells were stimulated with TZ, and extracellularly released and intracellular HMGB1-mCherry were analyzed every 2 min. Representative images of extracellularly released (TIRF) and intracellular (Epi) HMGB1-mCherry at the indicated times (d). Epi indicates merged images of intracellular HMGB1-mCherry and bright field. Time 0 indicates the time when the extracellular release of HMBG1-mCherry started. Scale bar, 25 μm. Representative plots of the change in intensities of extracellularly released HMGB1-mCherry in a burst-mode cell and a sustained-mode cell (e). The duration of the HMGB1-mCherry release in individual cell (f). The duration of the HMGB1-mCherry release of a single cell is plotted and clustered into two groups. Centers of each group are 109 and 7.1 min, respectively. Temporal relationship of a decrease in intensities of total and nuclear HMGB1-mCherry and an increase in extracellularly released HMGB1-mCherry (g). Intensities of each fraction of HMGB1-mCherry were calculated by epi-fluorescence (left vertical axis) microscopy and TIRF (right vertical axis) and plotted at the indicated times. Representative plots of a burst-mode cell (left, n = 10 cells) and a sustained-mode cell (right, n = 8 cells) in the same experiment. All results are representative of at least two independent experiments