Fig. 2: IFNγ synergizes with TKI to induce GSDME-mediated pyroptosis of HCC.

a The content of LDH released from SNU-387 cells primed with IFNγ (10 ng/mL) for 24 h, followed by sorafenib (15 μM) for 24 h. b LDH released from Hep3B cells treated with IFNγ and sorafenib in the presence of cell death inhibitors including z-VAD (100 μM), IDN (20 μM), Nec-1(40 μM), or Fer-1 (10 μM). c Cell death of SNU-182 cells primed with IFNγ and then treated with sorafenib (15 μM) plus IDN (40 μM) for 48 h. d Representative images showing pyroptosis morphology with large bubbles from the plasma membrane in Hep3B cells treated with IFNγ and sorafenib. e Immunoblots of human PARP, caspase 3, and GSDME in IFNγ-primed Hep3B cells treated with sorafenib (8 μM) for 24 or 36 h. α-Tubulin served as a loading control. f Immunoblots of human cleaved-PARP, cleaved-caspase3, and GSDME in SNU-387 cells primed with IFNγ followed by sorafenib (15 μM) plus IDN (40 μM) for 24 h. g Cell death of SNU-182 cells primed by IFNγ, followed by sorafenib (15 μM) in the presence of caspase 3 inhibitor Z-DEVD-FMK (40 μM) for 48 h. h LDH released from WT or GSDME-deficient SNU-182 cells treated with IFNγ and sorafenib (12 μM) for 24 h. i, j Hep-55.1C cells stably expressing Empty or GSDME-Flag were treated with a combination of mIFNγ (10 ng/mL) and sorafenib (20 μM). GSDME cleavage was determined by immunoblotting (i), and LDH content in the culture medium was quantified (j). k Tumor growth of Empty or GSDME-Flag expressing Hep-55.1C cells inoculated in C57BL/6 mice that were treated with vehicle control or sorafenib (10 mg/kg). n = 6–7 mice/group. l, m Empty or GSDME-Flag expressing Hepa1-6 cells were inoculated in C57BL/6 mice and treated with vehicle control or sorafenib (10 mg/kg). Tumor growth was monitored over time for each group (l). Subcutaneous tumors were surgically removed and presented (m) at the endpoint. n = 10 mice per group. Data were shown as mean or mean ± SEM. p-values were calculated by one-way ANOVA (g) and two-way ANOVA (a–c, h, j–l).