Human ZBP1 induces cell death-independent inflammatory signaling via RIPK3 and RIPK1 (2022)
Figure 1

Figure 1 - ZBP1 stimulates inflammatory signaling independently of cell death

A. Schematic illustration of WT and Zα1α2-mutant (Zα1α2mut) human ZBP1 inducibly expressed in HT29 / TO-ZBP1 cells.

B. Western blot analysis of the dose-dependent expression of ZBP1 in HT29 / TO-ZBP1WT and HT29 / TO-ZBP1Zα1α2mut cells treated with indicated concentrations of Dox for 24 h. Blots are representative of three biological repeats.

C. Flow cytometry analysis of ZBP1 expression in HT29 / TO-ZBP1 cells treated with Dox for 24 h. FLAG antibody staining was used to determine ZBP1-expressing cells. Data is presented as mean with S.E.M (n = 5 biological replicates). Two-way ANOVA and Tukey's multiple comparisons tests were used to test for statistical differences between indicated condition and DMSO-treated condition within each cell line. **** p<0.0001 for all conditions.

D, E. Cell death analysis of HT29 and HT29 / TO-ZBP1 cells as indicated following 24 h treatment with Dox using SytoxGreen to stain dead cells. Data is presented as mean ± S.E.M ((D) n = 5 biological replicates, (E) n = 3 biological replicates). Two-way ANOVA and Sidak's multiple comparisons tests were used to test for the statistical differences between indicated condition and untreated condition within each cell line. n.s. = not significant (p ≥0.05); *** p = 0.0004; **** p < 0.0001.

F. Relative viability of HT29 / TO-ZBP1WT cells treated with 500 ng/mL Dox for up to 3 days in combination with 20 µM zVAD, 10 µM Nec1s, 10 µM GSK'872 and/or 1 µM NSA as indicated was determined by the CellTitre-Glo assay. Values are normalized to untreated wells. Data is plotted as mean with S.E.M. (n = 4 biological replicates). Two-way ANOVA and Dunnet's multiple comparisons tests were used to test for statistical differences between indicated condition and DMSO-treated condition. n.s. = not significant (p > 0.99); *** p = 0.0006 for zVAD, p = 0.0007 for zVAD+NSA; **** p<0.0001.

G. Cell death analysis of HT29 / TO-ZBP1WT Clone B9 cells following treatment as indicated using SytoxGreen to stain dead cells. Cells were pretreated with 100 nM LCL161, 20 µM zVAD combined with10 µM GSK'840, 10 µM GSK'872, or 10 µM Nec1s for 1 h followed by treatment with 10 ng/mL TNF. Cell death analysis was carried out at the end of 24 h incubation with all compounds. Data is plotted as mean with S.E.M. (n = 3 biological replicates). Repeated Measures one-way ANOVA and Sidak's multiple comparisons tests were used to test for statistical differences between indicated condition and untreated control. **** p < 0.0001; n.s. = not significant (p > 0.4).

H. Cytokine concentration in the culture media of HT29 / TO-ZBP1 cells treated for 24 h with the indicated Dox concentrations or vehicle. Data is presented as mean with S.E.M (n = 4 biological replicates). Two-way ANOVA and Sidak's multiple comparisons t-test were used to test for the statistical differences between the two cell lines at each concentration. n.s. = not significant (p > 0.5); ** p = 0.0035 for CXCL8 at 20 ng/mL, p = 0.0067 for CXCL1 at 20 ng/mL, p = 0.0020 at 50 ng/mL, p = 0.0013 at 40 ng/mL; *** p = 0.0004 for CXCL8 at 25 ng/mL, p = 0.0008 at 30 ng/mL; **** p < 0.0001.

I. Western blot analysis of cells from the same wells as used in (H) for ZBP1 expression levels, representative of four biological replicates.

J. CXCL8 concentration in the culture media from the experiment described in (E). Data is presented as mean with S.E.M. (n = 3 biological replicates). Two-way ANOVA tests and Sidak's multiple comparisons test were used to test for statistical differences between indicated conditions. ** p = 0.0079; **** p < 0.0001.

K. Transwell migration of THP1 cells towards conditioned media from HT29 / TO-ZBP1WT cells treated with DMSO or 500 ng/mL Dox for 24 hours. Data is presented as mean with S.E.M. (n = 3 biological replicates of conditioned media). An unpaired t-test was used to test for statistical differences between indicated conditions. **** p < 0.0001.

L. Transwell migration of primary neutrophils towards conditioned media from HT29 / TO-ZBP1WT cells treated with DMSO or 500 ng/mL Dox for 24 hours, or HT29 chemotaxis buffer (control buffer) containing equal volume and amount of DMSO or Dox as the conditioned media. Data is presented as individual values with grand mean of the migrated percentage induced by control buffer containing DMSO or Dox (n = 10 biological replicates), or conditioned media from DMSO-treated cells (n = 11 biological replicates) or Dox-treated cells (n = 12 biological replicates), where the number of biological replicates is defined by the total number of independent cell cultures tested on primary neutrophils from four donors. An unpaired t-test was used to test for statistical differences between indicated conditions. **** p < 0.0001.

M. Transwell migration of primary neutrophils towards conditioned media from HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox in combination with DMSO or 20 µM zVAD + 10 μM Nec1s for 24 h. Data is presented as individual values with mean and S.E.M. (n = 3 biological replicates of conditioned media). One-way ANOVA and Sidak's multiple comparisons test were used to test for statistical differences between indicated conditions. ns = not significant (p = 0.4129); *** p = 0.0004.