Fig. 6: The in vivo antitumor activity of OSW-1 is mediated by p53/PUMA/MLKL-dependent necroptosis.

A–D C57BL/6 mice were injected s.c. with 5 × 10⁵ WT, p53-KO, or PUMA-KO MC38 cells (n = 9–10 in each group). After 7 days, mice were treated with OSW-1 (i.p.; 12.5 μg/kg) every other day as indicated by arrows. A Tumor volume at indicated time points after treatment with statistical significance for indicated comparisons. B Representative tumors at the end of the experiment. Scale bars, 10 mm. C H&E staining of indicated organ tissues. Scale bars, 100 μm. D Western blotting of indicated proteins. E Paraffin-embedded tumor tissues from mice treated as in (A) and resected at day 11 were analyzed by immunostaining for CD8. Left, representative staining pictures with arrows indicating example cells with positive staining (Scale bars, 20 μm); right, quantification of CD8⁺ cells (n = 5 in each group). At least 300 nuclei from 3 randomly selected fields were counted for each tumor. F–H C57BL/6 mice were injected with WT or MLKL-KO MC38 cells and treated with OSW-1 as in (A) (n = 7 in each group). F Tumor volume at indicated time points after treatment with statistical significance for indicated comparisons. G Representative tumors at the end of the experiment. Scale bars, 10 mm. H Western blotting of indicated proteins. In (D, H), pooled lysates from three randomly selected tumors in each group were analyzed. **, P < 0.01; ***, P < 0.001.