Figure 5

⍺-hemolysin induces tumor cell lysis in vitro and stimulates the cleavages of Caspase-3 and GSDME. (A) The dose–response curve of the viability of EO771 (left panel) and 4T1 tumor cells (right panel) that were treated with 5 to 200 μg/ml of HLA for 18 h (n = 3). (B) The viability of EO771 (left panel) and 4T1 cells (right panel) that were treated for 0, 2, and 4 h with 200 μg/ml and 100 μg/ml of HLA, respectively. (C) The levels of lytic cell death were measured by the percentage of LDH release in culture media of EO771 (left panel) and 4T1 cells (right panel) that were treated for 0, 2, and 4 h with 200 μg/ml and 100 μg/ml of HLA, respectively. (D) The representative bright-field microscopic images of EO771 cells treated with 200 μg/ml of HLA or vehicle control (Con) for 90 min (left) and 4T1 cells treated with 100 μg/ml of HLA or vehicle control (Con) for 2 h (right). Scale bars are shown in white at 20 μm. (E) EO771 cells were treated with 200 μg/ml of HLA for 0, 1, 2, and 4 h and the protein expression of cleaved GSDME and cleaved Caspase-3 were analyzed by Western Blot. Original blots are presented in Supplementary Information Source data 1. (F) The dose–response curve of the viability of MCF10A (in blue) and BT549 cells (in red) that were treated with 0.01 to 5 μg/ml of HLA for 18 h (n = 3 for 0.01–2 μg/ml, n = 2 for 5 μg/ml). Data are presented as mean ± s.e.m. One-way analysis of variance (ANOVA) with multiple comparisons ((B) and (C)). ***P < 0.001; ****P < 0.0001.