Fig. 4

HOCl promotes translocation and secretion of disulfide HMGB1 from PC12 cells. The PC12 cells were treated with HOCl (25, 50, 100 µM, dissolved in HBSS) for 15 min after exposure to 4 h OGD or normoxic conditions (NC), and then incubated under NC for 24 h. Taurine (2 mM) was added at the onset of HOCl exposure. A Western blot revealing the expression of HMGB1 in PC12 cells and the supernatant at 24 h of re-incubation under NC conditions. B Quantitative analysis of HMGB1 in the cells. C Western blot revealing the expression of HMGB1 in PC12 cells and the supernatant after treatment of 4 h OGD plus 24 h reoxygenation. D Quantitative analysis of HMGB1 in the cells. E, F Annexin V-FITC/PI staining flow cytometry detection for apoptotic cell death by using apoptotic detection kit. G Immunofluorescent detection for HMGB1 location in the PC12 cells with or without HOCl treatment. H Quantitative analysis of HMGB1 level in nuclear with or without HOCl treatment. I Quantitative analysis of HMGB1 level in the cytoplasm with or without HOCl treatment. J Western blot showing the fully reduced HMGB1 (fr-HMGB1) and disulfide-HMGB1 (ds-HMGB1) in cytoplasm and supernatant of the PC12 cells after 4 h OGD plus 24 h reoxygenation. K Quantitative analysis of ds-HMGB1 in the cytoplasm. L ELISA detection of HMGB1 secretion in the supernatant of PC 12 cells after 4 h OGD plus 24 h reoxygenation. *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01. All data are presented as mean ± SEM. (Statistical methods: B, H, I two-tailed t-test; D, K, L one-way ANOVA followed by Tukey’s multiple comparisons test.)