Fig 2: Irradiation in EC cell lines induces caspase-3 and GSDME cleavage. (A–E) ECA-109 and KYSE-150 cells were exposed to 0, 2, 4, and 8 Gy of X-ray. The LDH release of ECA-109 (A) and KYSE-150 cells (B) was analyzed using an LDH detection kit. The expression of caspase-3 and GSDME in ECA-109 (C) and KYSE-150 cells (D) was detected by western immunoblotting. (E) Morphology of ECA-109 and KYSE-150 cells was observed under a light microscope. n.s., not significant; *p < 0.05; **p < 0.01; and ***p < 0.001 vs control.

Fig 3: Overexpression of caspase-3 and GSDME aggravates irradiation-induced ICD. (A–H) ECA-109 cells were transfected with caspase-3 or GSDME overexpression plasmids and then exposed to 8 Gy of X-ray. The expression of caspase-3 (A) and GSDME (B) in ECA-109 cells was measured by using western immunoblotting. Apoptosis of ECA-109 cells following transfection with caspase-3 (C) and GSDME (D) overexpression plasmids were analyzed using an annexin V-FITC/PI detection kit. The LDH release of ECA-109 cells followed transfection with caspase-3 (E) and GSDME (F) overexpression plasmids using an LDH detection kit. Expression of HSP70 and HSP90 in ECA-109 following transfection with caspase-3 (G) and GSDME (H) overexpression plasmids were measured by western immunoblotting. *p < 0.05; **p < 0.01; and ***p < 0.001 vs the indicated group.

Fig 4: Silencing of caspase-3 reverses irradiation-induced GSDME cleavage. (A–D) ECA-109 and KYSE-150 cells were transfected with caspase-3 specific siRNA and then exposed to 8 Gy of X-ray. The LDH levels in ECA-109 (A) and KYSE-150 cells (B) were measured using an LDH detection kit. The expression of GSDME in ECA-109 (C) and KYSE-150 cells (D) was measured using western immunoblotting. *p < 0.05; **p < 0.01; and ***p < 0.001 vs the indicated group.

Fig 5: Silencing of GSDME reverses irradiation-induced tumor cell death. (A–F) ECA-109 and KYSE-150 cells were transfected with GSDME-specific siRNA and then exposed to 8 Gy of X-ray. The expression of GSDME in ECA-109 (A) and KYSE-150 cells (B) was measured by using western immunoblotting. The apoptosis of ECA-109 (C) and KYSE-150 cells (D) was analyzed using an annexin V-FITC/PI detection kit. The LDH levels in ECA-109 (E) and KYSE-150 cells (F) were measured using an LDH detection kit. n.s., not significant; *p < 0.05; **p < 0.01; and ***p < 0.001 vs the indicated group.