Fig. 6

SZP induces autophagy-dependent ICD in vitro. A Schematic diagram of SZP inducing ICD. B Reduced expression of ATG5 in HCC cells with ATG5 knocked down. C, E Immunoblotting and ELISA of HMGB1 in the supernatant of sublethally heated HCC cells (SMMC7721 and Huh7) and ATG5-knockdown HCC cells treated with SZP (n = 4). D The concentration of ATP within and outside the sublethally heated HCC cells and ATG5-knockdown HCC cells treated with SZP (n = 5). F Representative CLSM graphs of the CRT exposure and HMGB1 release of sublethally heated HCC cells and ATG5-knockdown HCC cells treated with SZP. Scale: 30 µm. G Schematic diagram of transwell co-culture. Sublethally heated HCC cells and ATG5-knockdown HCC cells treated with SZP (upper chamber) and BMDCs (lower chamber) were co-cultured for 24 h. H, I Representative flow cytometry plots and quantitative analysis of the maturation of DCs (of CD11C⁺ gate) (n = 3). J The levels of IL-12p70, IL-1β, TGF-β, and IL-6 in the supernatants of co-cultured DCs measured by ELISA (n = 4). Results were expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. ICD, Immunogenic death; CRT, Calreticulin; BMDCs, Bone marrow-derived DC cells