Fig. 9

Cinobufagin Induces Immunogenic PANoptosis and Potentiates GBM to Anti-PD-1 therapy. A Propidium iodide (PI) staining showing the death of G003 and G007 cells treated with cinobufagin (CB), pyroptosis inhibitor (Belnacasan), apoptosis inhibitor (V-ZAD-FMK), and necroptosis inhibitor (Necrostatin) for 24 h. B Multiplex immunohistochemistry (mIHC) of N-GSDME, c-CASP3 and p-MLKL in G003 cells treated with CB for 24 h. C Western blot (WB) analysis of PANoptosis markers (CASP3/7, c-CASP3/7, GSDMD, GSDME, MLKL, and p-MLKL) in GBM cell lines and primary GBM cells. D-F Flow cytometry showing mitochondrial stress markers in G003 and G007 cells following CB treatment: (D) MitoSox-red for mitochondrial superoxide, (E) DCFH-DA for intracellular ROS, and (F) MitoTracker for mitochondrial mass. G Flow cytometry analysis of the externalization of calreticulin to the cell membrane (ExoCRT). H Release of extracellular ATP quantified via an ADP/ATP ratio assay kit. I Schematic of the in vivo experimental design using GL261 and CT2A cells in C57BL/6J mice. J-K In vivo imaging on day 15 postimplantation for GL261 and CT2A tumors. L-M Quantitative bioluminescence analysis for (J) and (K), respectively. N = 4–5 mice/group. N-O Kaplan-Meier survival curves for GL261 and CT2A tumor-bearing mice under different treatments. P-S Immunofluorescence (IF) showing infiltration of CD45⁺, CD4⁺, CD8⁺, and IBA1⁺ cells in CT2A tumors. N = 6–7 mice/group. T Flow cytometry of CD45⁺ cells in GL261 tumors. N = 6 mice/group. U-V Flow cytometry of CD4⁺/CD8⁺ T cells and F4/80⁺ cells in GL261 tumors, showing absolute infiltration and relative proportion among CD45⁺ cells