Fig. 5

Blockade of ROS and IRE1α/XBP1s axis inhibited ICD induced by DSF/Cu and irradiation in BCSCs. Sorted BCSCs were pretreated with 10 μM 4u8c, 10 μM STF-083010 or 10 mM NAC for 1 h. The inhibitor(s) containing medium was replaced with fresh culture medium with DSF/Cu (0.15/1 μM) and cultured for 24 h. Then DSF/Cu containing medium was removed and replaced with fresh culture medium and then irradiated at 12 Gy and cultured. The cells and/or culture supernatants were collected at indicated times post IR and used for analyses of ICD. (a) Apoptotic cells were quantitated 24 h post-IR (top panel). Percentages of apoptotic cells are indicated (bottom panel). (b) Percentages of cells expressing CRT were detected 8 h post IR (top panel) and their mean fluorescence intensity (MFI) values are shown (bottom panel). (c) Percentages of cells expressing HSP90 were detected 8 h post IR (top panel) and their MFI values are shown (bottom panel). (d) Levels of intracellular ATP were measured 1 h post-IR (top panel) and their MFI values are shown (bottom panel). (e) Concentrations of HMGB1 release were determined in supernatants collected at 24 h post-IR. *indicates p < 0.05, **indicates p < 0.01. These data demonstrate that blocking ROS generation and IRE1α/XBP1s signaling pathway partially abolished elevated ICD of BCSCs by IR and DSF/Cu and implies that both pathways are involved in the enhanced immunogenic apoptosis of BCSCs induced by this combinatorial approach