Human ZBP1 induces cell death-independent inflammatory signaling via RIPK3 and RIPK1 (2022)
Figure 5

Figure 5 - RIPK3 oligomerization induces ubiquitination-mediated inflammatory signaling.

A. Schematic illustration of the chemical-inducible RIPK3 oligomerization system.

B. Western blot analysis of HCT116 / RIPK3-2xFV cells (FL and ∆C) treated with 100 nM dimerizer. Blots are representative of two biological replicates.

C. Time courses of mRNA levels of CXCL8 and CXCL1 in HCT116 / RIPK3-2xFV cells (FL and ∆C) in response to treatment with 100 nM dimerizer. Data is plotted as mean with S.E.M. (n = 3 biological replicates). Two-way ANOVA and Tukey's multiple comparisons test were used to test for statistical differences between 0 h and indicated time points in FL cells. ns = not significant (p = 0.1360); ** p = 0.0046 for FL 2 h, p = 0.0012 for FL 3 h; **** p < 0.0001; *** p = 0.0008.

D. CXCL8 concentration in the culture media of HCT116 / RIPK3-2xFV cells (WT and RHIMmut) treated with 100 nM dimerizer for 24 h. In RIPK3-RHIMmut three key residues, VQV, of the RHIM region was changed to AAAA. Data is plotted as mean with S.E.M. (n = 5 biological replicates). One-way ANOVA and Sidak's multiple comparisons tests were used to test for statistical differences between indicated time points. ** p = 0.0011; ns = not significant (p = 0.9301). Inset: Western blot analysis to determine RIPK3 expression levels in one biological replicate. Line indicates that image was cut and spliced to remove non-relevant lanes from the scanned blot.

E. Relative viability of HCT116 / RIPK3-2xFV cells treated with 0 or 100 nM dimerizer for 24 h. Data is presented as mean with S.E.M. (n = 3 biological replicates). A Welch's t-test was used to test for statistical differences between indicated conditions. ns = not significant (p = 0.2294).

F. Cell death analysis of HCT116 / RIPK3-2xFV cells treated with 0 or 100 nM dimerizer or TSZ (100 nM CpA, 20 μM zVAD and 1 ng/mL TNF) for 24 h using SytoxGreen to stain dead cells. Data is plotted 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 difference between indicated conditions. n.s. = not significant (p = 0.1032); ** p = 0.0014.

G, H. Relative mRNA levels of CXCL8 and CXCL1 in HCT116 / RIPK3-2xFV cells pretreated with DMSO, TAK1 inhibitor, or IKK inhibitors for 1 h or 100 nM CpA for 0.5 h before treatment with 0 or 100 nM dimerizer for 3 h. 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 indicated conditions and DMSO+dimerizer-treated condition. **** p < 0.0001. (H) Cell lysates were analyzed by Western blotting for cIAP1 levels. Blots are representative of two biological replicates.

I. CXCL8 concentration in the culture media of HCT116 / RIPK3-2xFV cells pretreated with 100 nM CpA for 1 h before treatment with 0 or 100 nM dimerizer for 24 h. Data is presented as mean with S.E.M. (n = 3 biological replicates). An unpaired t-test was used to test for the statistical difference between indicated conditions. ** p = 0.0042.

J. Relative CXCL8 mRNA levels in WT or HOIP-knockout HCT116 / RIPK3-2xFV cells treated with 0 or 100 nM dimerizer for 3 h. Data is plotted as mean with S.E.M. (n = 3 biological replicates). A Welch's t-test was used to test for the statistical difference as indicated. *** p = 0.0010. Cell lysates were loaded for Western blot analysis.

K. Relative CXCL8 mRNA levels in HCT116 / Tet-On-GFP-K63-SUB / RIPK3-2xFV, HCT116 / Tet-On-GFP-M1-SUB / RIPK3-2xFV and HCT116 / Tet-On-GFP / RIPK3-2xFV cells treated with 0 or 100 ng/mL Dox for 48 h before stimulated with 0 or 100 nM dimerizer for 3 h. Data is presented as mean with S.E.M. (n = 4 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. ns = not significant (p = 0.9997); ** p = 0.0030; * p = 0.0366. Cells were analyzed by Western blotting for the inducible-expression levels of GFP (control) and GFP-SUBs. Blots are representative of three biological replicates.

L. Relative chemokine mRNA levels in HCT116 / RIPK3-2xFV cells pretreated with 0 or 20 µM zVAD in combination with DMSO, 10 µM Nec1s, 10 µM GSK'872 or 1 µM NSA for 1 h, followed by treatment with 0 or 100 nM dimerizer for 3 h. Data is plotted as mean with S.E.M. (n = 3 biological replicates). **** p < 0.0001. One-way ANOVA and Sidak's multiple comparison test were used to test for statistical differences between indicated conditions.

M. Relative CXCL8 mRNA levels in HCT116 / RIPK3-2xFV cells stably knocked down against mismatch (shMM) or two different sites of caspase-8 (shCASP8-A and shCASP8-B), pretreated with DMSO, 10 µM Nec1s, 10 µM GSK'872 or 1 µM NSA for 1h followed by treatment with 0 or 100 nM dimerizer for 3h. Data is plotted as mean with S.E.M. (n = 3 biological replicates). Two-way ANOVA and Tukey's multiple comparison tests were used to test for statistical differences between indicated conditions. *** p = 0.0001; ** p = 0.0035. Cell lysates from one biological replicate were analyzed by Western blotting to determine caspase-8 levels.