RIPK3-MLKL pathway prevents systemic spread of Listeria in mice. (A) Human expression levels by tissue for RIPK3. Gene-level transcripts per kilobase million (TPM) were downloaded from the GTEx portal and summarized across tissue type. Box and whisker plots are plotted in order of highest median expression to lowest. The bottom border, middle line, and top border of the box represent the first quartile, second quartile (median), and third quartile, respectively, while the lines represent 150% of the interquartile range. Outliers more than 150% of the interquartile range are represented as points. 17 representative tissues are shown (see Fig. S1 A for the full results). (B and C) Protein extracts of the indicated tissues from Ripk3^+/+ (WT, #1–3) and Ripk3^−/− (knockout [KO], #1) mice were analyzed by Western blotting with the indicated antibodies. Coomassie brilliant blue (CBB) staining of the whole membrane (B) and β-actin (C) were used as loading controls. (D and E) Ripk3^−/− (KO) and littermate Ripk3^+/+ or Ripk3^+/− (control) mice were orally infected with Listeria. Immunofluorescence staining of liver-colonized Listeria (D) and CFU of Listeria in the liver (E) at 3 d after infection are shown. Arrowheads indicate Listeria in the liver. Bar, 50 µm (mean ± SEM; 6–9-wk-old; control, n = 9; Ripk3 KO, n = 9; *, P < 0.05). (F) Mlkl^−/− (KO) and littermate Mlkl^+/+ (control) mice were orally infected with Listeria. CFU of Listeria in the liver at 3 d after infection is shown (mean ± SEM; 6–9-wk-old; control, n = 9; Mlkl KO, n = 4; *, P < 0.05).