Fig. 1

Pyroptosis is induced via the NLRP3/caspase-1/GSDMD pathway when KLE and HEC-1A cells are treated with DDP. A KLE and HEC-1A cells were treated with DDP at various concentration gradients (0, 5, 10, 20, 40, and 80 µg/mL) for 48 h; then, cell viability was assessed using the CCK8 assay. B, C The pyroptotic cells (PI-positive) were imaged using fluorescence microscopy after DDP treatment for 12 h (the values in each graph represent the average of three random fields per sample); scale bar: 100 µm. The KLE and HEC-1A cells from the control group were treated with the vehicle 0.9% NaCl. D, E The percentages of pyroptotic Annexin V-PE- and 7-AAD-positive cells within the second quadrant (Q2) were determined using flow cytometry after treating KLE and HEC-1A cells with DDP for 24 h. CON indicates the control group of KLE and HEC-1A cells treated with the vehicle 0.9% NaCl. F Representative TEM images of KLE and HEC-1A cells treated with DDP for 12 h. The mitochondrial membrane was disrupted, the matrix was dissolved, and the mitochondrial cristae were broken, as indicated with red arrows. The control group KLE and HEC-1A cells were treated with the vehicle 0.9% NaCl; the yellow arrows indicate the mitochondria structures that were more complete. Scale bar: 2 μm. G LDH activity in the culture supernatants of KLE and HEC-1A cells treated with DDP for 12 h was measured. H, I Whole-cell lysates of KLE and HEC-1A cells treated with DDP for 12 h or left untreated were analyzed using WB. The control group KLE^−vehicle and HEC-1A^−vehicle cells were treated with 0.9% NaCl. β-Actin was included as a loading control. The dose of DDP in the above-mentioned in vitro experiments was 7 µg. Data are representative of three independent experiments and shown as the mean ± SD. *p < 0.05; **p < 0. 01; ***p < 0.001; ****p < 0.0001. Abbreviations: CCK8, Cell Counting Kit-8; PI, propidium iodide; TEM, transmission electron microscopy; LDH, lactate dehydrogenase; WB, western blotting