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Fig. 4 | Cell Communication and Signaling

Fig. 4

From: Induction of immunogenic cell death in radiation-resistant breast cancer stem cells by repurposing anti-alcoholism drug disulfiram

Fig. 4

DSF/Cu rendered BCSCs as sensitive as non-BCSCs to IR-induced ICD. Sorted sells were pretreated with DSF/Cu (0.15/1 μM) for 24 h, then DSF/Cu containing medium was removed and replaced with fresh culture medium and then irradiated at 8 or 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 (left panel). Percentages of apoptotic cells are indicated (right panel). (b) Cleaved PARP, an indicator of apoptosis, was detected in both BCSCs and non-BCSCs treated by DSF/Cu and IR. (c) Percentages of cells expressing CRT were detected 8 h post IR (left panel) and their mean fluorescence intensity (MFI) values are shown (right panel); Percentages of 7AAD+ cells expressing CRT were detected 24 h post IR (left panel) and means ± SD of 7AAD+CRT+ cells (%) are shown (right panel). (d) Percentages of cells expressing HSP90 were detected 8 h post IR (left panel) and their MFI values are shown (right panel); Percentages of 7AAD+ cells expressing HSP90 were detected 24 h post IR (left panel) and means ± SD of 7AAD+HSP90+ cells (%) are shown (right panel), PE-conjugated rabbit isotype control mAb was simultaneously stained cells with PE-conjugated rabbit mAb for CRT and HSP90 24 h post IR-negative control results shown as C, D isotype control. (e) Levels of intracellular ATP were measured 1 h post-IR (left panel) and their MFI values are shown (right panel); Levels of extracellular ATP release were measured 24 h post-IR. (g) Concentrations of HMGB1 release were determined in supernatants collected at 24 h post-IR. (g) CRT expression was quantitated in dead (7-AAD+) and live (7-AAD) MDA-MB-231 BCSCs. *indicates p < 0.05, **indicates p < 0.01

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