Fig 2: XIAP BIR1- and -BIR2 support melanoma growth via TAB1- and RIPK2-dependent signalling B16F1 WT, B16F1-XIAP?BIR1 and B16F1-XIAP?BIR2 subcutaneous melanoma tumour growth in BL/6 WT mice (n = 3 each genotype).B16F1 WT, B16F1-XIAPKO (clone 1 and 2), B16F1-TAB1KO and B16F1-RIPK2KO (clone 1 and 2) subcutaneous melanoma tumour growth in BL/6 WT mice (n = 5 each genotype).qRT–PCR analysis of different cytokines/chemokines in the indicated melanoma tumours in mice. Dots represent individual mice (n = 3). Data are presented as mean ± SD. One-way analysis of variance (ANOVA) followed by Dunnett’s post-analysis. Data information: In A and B data are mean ± s.e.m; two-way analysis of variance (ANOVA) followed by Dunnett’s post-analysis. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 and ns, not significant.

Fig 3: XIAP BIR1- and -BIR2 support melanoma growth via TAB1- and RIPK2-dependent signalling Western blot analysis of cell lysates of B16F1 WT, B16F1-XIAPKO (clone 1 and 2), B16F1-XIAP?BIR1 (clone 1 and 2), and B16F1-XIAP?BIR2 (clone 1 and 2) after CRISPR–Cas9 gene editing.mRNA expression level of Xiap in B16 melanoma cells. Dots represent individual biological replicates. Data are presented as mean ± SD.Western blot analysis of cell lysates of B16F1 WT, B16F1-XIAPKO (clone 1 and 2), B16F1-TAB1KO and B16F1-RIPK2KO (clone 1 and 2) after CRISPR–Cas9 gene editing.Representative fluorescent images of cl-Caspase 3+ cells in B16F1 WT, XIAPKO, TAB1KO or RIPK2KO melanoma tumours. Nuclei were stained with DAPI (blue). Scale bar 100 µm. Quantification thereof (lower panel). The average of cl-Caspase 3 positive cells from 5 randomly selected areas of the tumour (5 mice per genotype) was represented. Dots represent individual mice (n = 5). Data are presented as mean ± s.e.m.Cell death measurement of B16F1 WT, TAB1KO and RIPK2KO (clone 1 and 2) cells using Incucyte analysis after 24 h treatment with DMSO as a negative control or 100 ng/ml TNF. H2O2 treatment serves as a positive control. Scale bar 200 µm.Proliferation assay using Incucyte Live-Cell-Analysis assay. Data information: In B, and D one-way analysis of variance (ANOVA) followed by Dunnett’s post-analysis. In E and F, data are presented as mean ± s.e.m; two-way analysis of variance (ANOVA) followed by Tukey’s post-analysis. Data derived from three individual biological replicates. ****P < 0.0001 and ns, not significant. Source data are available online for this figure.

Fig 4: XIAP mediates IL8 secretion in melanoma by involving TAB1 and RIPK2 AIL8 measurement in the supernatant of transfected HEK293T cells after 24 h. Western blot analysis of cell lysates of HEK293T cells transfected with the indicated plasmids (bottom).BWestern blot analysis of cell lysates (input) and myc-IP of HCT116 cells transfected with pcDNA as a negative control or myc-XIAP for 24 h.CConfocal microscopic analysis of HeLa cells transfected with the indicated plasmids for 16 h and stained for TAB1 or RIPK2 (red). Nuclei were stained with DAPI (blue). Scale bar 20 µm.DIL8 measurement in the supernatant of transfected HCT116 WT, XIAPKO, RIPK2KO and TAB1KO cells after 24 h. One-way analysis of variance (ANOVA) followed by Sidak’s post-analysis. Western blot analysis of cell lysates of the respective cell lines transfected with the indicated plasmids (bottom).E, FIL8 measurement in the supernatant of transfected HCT-RIPK2KO (E) or HCT-TAB1KO (F) cells after 24 h. Western blot analysis of cell lysates of HCT-RIPK2KO and HCT-TAB1KO cells transfected with the indicated plasmids.GIL8 measurement in the supernatant of BLM and SK-Mel28 after siRNA transfection (48 h).HIL8 measurement in the supernatant of HCT116 cells transfected with the indicated siRNA for 48 h and treated with DMSO (control) or MDP for 24 h. Data information: In A, D, E, F, G, and H, data are presented as mean ± SD. In A, E, F, G and H one-way analysis of variance (ANOVA) followed by Dunnett’s post-analysis. In A, D, E, F, G, and H, dots represent individual biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 and ns, not significant. Source data are available online for this figure.

Fig 5: XIAP mediates IL8 secretion in melanoma by involving TAB1 and RIPK2 Western blot analysis of cell lysates of BLM and BLM-XIAPKO cells (input) and XIAP-IP. Data are representative of two experiments.Confocal microscopic analysis of transfected HCT116 cells with GFP-XIAP for 16 h and stained for TAB1 or RIPK2 (red). Nuclei were stained with DAPI (blue). Scale bar 20 µm.Cell-free ubiquitylation assay of recombinant GST-RIPK2 by Flag-XIAP after addition of respective E1/E2 and recombinant ubiquitin. Poly-ubiquitylated RIPK2 and XIAP were detected by Western blotting.Western blot analysis of cell lysates of BLM, BLM-XIAPKO, SK-Mel28, SK-Mel28-XIAPKO, B16, and B16-XIAPKO cells (input) and RIPK2-IP. Poly-ubiquitin is detected using anti-ubiquitin antibody.IL8 measurement in the supernatant of transfected BLM (left panel) or SK-Mel28 (right panel) cells with siScr (control), siXIAP, siTAB1 or siRIPK2 after 48 h. Western blot analysis of the respective cell lysates of BLM cells (left bottom) or SK-Mel28 cells (right bottom) transfected with the indicated siRNA.IL8 measurement in the supernatant of BLM or SK-Mel28 treated with 2 µM d89 (BIR2 antagonist) or DMSO as a negative control for 24 h.IL8 measurement in the supernatant of BLM or SK-Mel28 treated with 20 µM birinapant (Bir), 2 µM BV6 (both BIR3 antagonist) or DMSO as a negative control for 24 h. Data information: In E, F and G, dots represent individual biological replicates. Data are presented as mean ± SD. Unpaired t-test, two-tailed test analysis (F) or One-way analysis of variance (ANOVA) followed by Dunnett’s post-analysis (E and G). *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001. Source data are available online for this figure.