Human ZBP1 induces cell death-independent inflammatory signaling via RIPK3 and RIPK1 (2022)
Figure 2

Figure 2 - ZBP1-induced inflammatory signaling requires RIPK3 and RIPK1 but not their kinase activity

A. Cytokine concentration in the culture media of HT29 / TO-ZBP1WT cells treated with 0 or 50 ng/mL Dox in combination with indicated inhibitors for 24 h. TAK1i, 1 μM 5z-7-oxozeaenol. IKKi, 1 μM IKK Inhibitor VII + 5 μM IKK Inhibitor XII. Nec1s, 10 μM. GSK'872, 10 μM. NSA, 1 μM. Data is presented as mean with S.E.M. (n = 3 biological replicates). One-way ANOVA and Sidak's multiple comparisons tests were used to test for statistical differences between Dox-induced samples pretreated with DMSO and corresponding inhibitor. ns = not significant (p > 0.2); * p = 0.0104; ** p = 0.0041; **** p < 0.0001.

B. Relative viability of HT29 / TO-ZBP1WT cells treated with the same conditions as in (A). Data is plotted as mean with S.E.M. (n = 4 biological replicates). One-way ANOVA and Sidak's multiple comparisons tests were used to test for statistical differences between all conditions and DMSO-treated condition, with significantly different conditions indicated. * p = 0.023; *** p = 0.0002.

C. Transwell migration of primary neutrophils towards conditioned media from HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox in combination with indicated inhibitors for 24 h. Data is presented as individual values with grand mean (n = 3 biological replicates of conditioned media from GSK'872 and NSA-treated wells, n = 6 biological replicates of conditioned media from other conditions). Two-way ANOVA and Sidak's multiple comparisons test were used to test for statistical differences between indicated conditions. **** p < 0.0001; ns = not significant (p > 0.96).

D. CXCL8 concentration in the culture media of HT29 / TO-ZBP1WT cells transfected with siRNA targeting mismatch sequence (MM) or RIPK3 and treated with Dox at indicated concentrations for 24 h. Data is presented as mean with S.E.M. (n = 4 biological replicates). Unpaired t-tests were used to test for statistical differences between indicated conditions. ** p = 0.0079. Cell lysates from one biological replicate were analyzed by Western blotting.

E. Cytokine concentration in the culture media of HT29 / TO-ZBP1WT or HT29 / RIPK1-KO clone aA3 / TO-ZBP1WT cells treated with 0, 50 or 500 ng/mL Dox. data is plotted as mean with S.E.M. (n = 6 biological replicates). Brown-Forsythe and Welch ANOVA tests and Dunnet's T3 multiple comparisons test were used to test for statistical differences between indicated conditions. Cell lysates were analyzed by Western blotting for ZBP1 levels. Blots are representative of six biological replicates. ns = not significant (p > 0.2); *** p = 0.0003 for CXCL8, p = 0.0008 for CXCL1; **** p < 0.0001.

F. Relative viability of HT29 / TO-ZBP1WT and HT29 / RIPK1-KO clone aA3 / TO-ZBP1WT cells treated with 500 ng/mL Dox for up to 3 days. Values are normalized to day 0 of treatment within each cell line. Data is plotted as mean with S.E.M. (n = 3 biological replicates). Two-way ANOVA and Sidak's multiple comparison tests were used to test for statistical differences between WT and RIPK1-KO cells at the indicated time points after Dox treatment. *** p = 0.0003; **** p < 0.0001.

G, H. NF-κB activity in HEK293FT cells with and without stable expression of full-length (FL) RIPK3-2xFV or RIPK3∆C-2xFV (∆C) transfected with dual luciferase reporters and ZBP1 or empty vector (EV) as indicated. In (H), inhibitors were added immediately after transfection: Nec1s, 10 μM. GSK'872, 10 μM. NSA, 1 μM. Reporter activities were measured 24 h after transfection. Data is presented as mean with S.E.M (n = 3 biological replicates). One-way ANOVA and Sidak's multiple comparisons test were used to test for statistical differences between indicated conditions. ns = not significant (p > 0.05); **** p<0.0001. (G) Cell lysates were analyzed by Western blotting. Blots are representative of three biological replicates. Asterisk indicates background signals of the antibody.