Figure 4.

RIPK3 deficiency enhances thymic lymphoma in p53‐deficient mice via ERK hyperactivation. A) Kaplan‐Meier survival curve (Long‐rank test) of animals in different genetic backgrounds including Ripk3^+/+p53^−/− and Ripk3^−/−p53^−/− animals. Red dotted line shows median survival of Ripk3^+/+p53^−/− and Ripk3^−/−p53^−/‐ mice, which are significantly different (p < 0.0001). B) The thymic lymphoma free survival curve and thymic lymphoma incidence of p53^−/− and Ripk3^−/−p53^−/− animals until 150 days of age. The tumor‐free survival plot of Ripk3^+/+p53^−/− (n = 42) compared with Ripk3^−/−p53^−/‐ (n = 158) mice that are significantly different. C) Representative images of thymus, spleen and lymph node from tumor‐bearing Ripk3^+/+p53^−/− and Ripk3^−/−p53^−/− littermates. D) The phenotype of the thymic T cell populations (left panel). The thymic T cell from both Ripk3^+/+p53^−/− and Ripk3^−/−p53^−/− thymic lymphoma showed homogenous expansion in DP T cells but Ripk3^−/−p53^−/− DP T cells showed increased proliferation index (Ki‐67) (right panel). E) RNA sequencing analysis of up‐ and down‐regulated gene in Ripk3 ^−/− p53 ^−/− thymic lymphoma compared to p53 ^−/− thymic lymphoma. Analysis of up‐regulated gene shown the change in positive regulation of ERK signaling pathways. F) Total thymocytes from Ripk3^+/+ (n = 4) and Ripk3^−/− (n = 4) mice were stimulated with anti‐CD3 (1 µg mL⁻¹) and anti‐CD28 (2 µg mL⁻¹) or without for indicated time. Phosphorylation of ERK in DP T cell measured by Flow Cytometry. G) The protein expression levels of p‐ERK in normal thymus (Ripk3^+/−p53^+/− ; n = 3, Ripk3^−/−p53^+/+ ; n = 3) and thymic lymphoma (Ripk3^+/+p53^−/− ; n = 10, Ripk3^−/−p53^−/− ; n = 10) tissues were measured by Western blot analysis. Statistical analyses were performed using the two‐tailed unpaired Student t‐test or log rank test. p values below 0.05 were considered significant in the following manner: *p < 0.05, **p < 0.01, ***p < 0.001.