Fig. 4

DOC enhanced HMGB1 secretion through a positive feedback process. a mRNA expression levels of several cytokines in lung cancer cells were analyzed by qRT-PCR, and are shown as expression heatmap in control group vs DOC treated group. Heatmap color ranging from minimum (white) to maximum (blue) represents the relative gene expression level. b HMGB1 protein levels in lung cancer cells in control group vs DOC treated group assessed by immunofluorescence. Scale bars, 50 μm. c HMGB1 protein levels in lung cancer cells in control group vs DOC treated group assessed by ELISA. Data are given as means ± SEM. Significant differences between each group are indicated by **p < 0.0001 or *p < 0.05. d mRNA expression of caspase 3 and caspase 6 in control and DOC-treated A549 cells. Significance is noted as *p < 0.05. e HMGB1 protein levels in A549 cells is upregulated by incubation with DOC, and inhibited by the caspase3/6 inhibitor Z-DEVD-FMK. Significance is noted as **p < 0.0001. f, g Contents of ROS in A549 cells were evaluated by flow cytometry after treatment with DOC or combined with ROS inhibitor (GSH) or Z-DEVD-FMK, and the differences were significant. h The expression of HMGB1 were also analyzed by ELISA after treatment with DOC or combined with GSH. **p < 0.0001. i, j ROS production in A549 cells treated with recombinant protein HMGB1(rHMGB1) or combined with GSH, as detected by FSC. *p < 0.05. k Schematic diagram illustrating the proper release mechanism of HMGB1. The green arrow represents the relationship between DOC and HMGB1, and the blue arrow represents the relationship between HMGB1 and ROS. Data are given as means ± SEM. Significance is noted as **p < 0.0001, *p < 0.05. All of the experiments were independently repeated three times