Fig. 6: Phosphorylated GD-NT loses the ability to mediate pyroptosis and anti-tumor immunity in vivo.

A HEK293 cells were transfected with Flag-GD-NT-WT or its mutants. 24 hours later, phase-contrast images were taken (left panel), and cells were harvested for IB analysis with indicated antibodies (right panel). B Similar to (A), except that the cells were subjected to LDH-based Cytotoxicity Assay or ATP-based Cell Viability Assay. C E0771 cells expressing Dox-inducible GD-NT-WT, GD-NT-S46D or empty vector were treated with or without Dox (2 µg/ml). 48 hours later, cells were harvested and subjected to LDH-based Cytotoxicity Assay. D, E E0771 tumor allograft in C57BL/6 Rag-1^-/- mice. E0771 cells expressing Dox-inducible GD-NT-WT or GD-NT-S46D (0.1 × 10⁶ cells per mouse) were implanted into 4^th mammary fat pad of Rag-1^-/- mice (n = 6 mice per group). Dox (50 mg/kg, i.p.) was administrated on day (d) 2 post-implantation and the following every other day. Tumor growth was recorded every other day and tumor weight was measured after sacrificing mice (E). F–H, GD-NT initiates anti-tumor immunity. 0.9 × 10⁶ of E0771 parental cells mixed with 0.1 ×10⁶ of the modified E0771 cells expressing GD-NT-WT or GD-NT-S46D were implanted into 4^th mammary fat pad in Rag-1^-/- mice (n = 4 mice per group) or naïve wild-type mice (n = 6 mice per group). Dox (50 mg/kg, i.p.) was administrated on d6 and d8 post-implantation. Tumor growth was recorded every other day (F and G). hematoxylin and eosin (H&E) staining of representative tumors at d20 was shown (H). In B, C, D, E, F and G, error bars represent the variation range of duplicated experiments. In B and E, differences among groups were analyzed by two-tailed Student`s t-test (means ± s.e.m). In D, F and G, the areas under the growth curves were compared by two-tailed Student`s t-test (means ± s.e.m). Data are representative of at least two independent experiments.