Fig. 10

Knockdown of Chmp4b abrogates a sustained-mode of HMGB1 release. a L929-SMART/HMGB1-mCherry cells were transfected with control or Chmp4b siRNA, and knockdown efficiency was determined by qPCR at 24 h after transfection. Results are means ± s.d. of triplicate samples and representative of two independent experiments. Statistical significance was determined using the unpaired two-tailed Student-t test. **P < 0.001. b Duration of the HMGB1-mCherry release of a single cell. Cells were treated as in (a) and then stimulated with TZ. The release of HMGB1 was analyzed as in Fig. 8 and the duration of the release was determined (n = 29 cells for control siRNA and n = 26 cells for Chmp4b siRNA). Centers of each group of cells treated with control siRNA are 144 and 4.4 min, whereas that of Chmp4b siRNA is 2.9 min. Each red dot indicates individual cell showing a sutained-mode of HMGB1 release. Results are representative of two independent experiments. Statistical significance was determined using the Mann–Whitney test. **P < 0.001. c, d Cells were treated with siRNAs and stimulated with TZ. The times between the start of an increase in ΔFRET/CFP ratio and the burst of cells were calculated and plotted (n = 10 cells for each treatment) (c). Pooled results of two independent experiments. Statistical significance was determined using the Mann–Whitney test. **P < 0.001. Kinetics of averages of ΔFRET/CFP ratio of a single cell was plotted (n = 10 cells for control or Chmp4b siRNA) (d). Time 0 indicates the start of an increase in ΔFRET/CFP ratio. Error bars indicate s.e.m.