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. 2021 Oct 8;12:5913. doi: 10.1038/s41467-021-25944-2

Fig. 3. Dermatitis in ΔKerOTULIN mice depends on TNFR1-signaling and on FADD/MLKL- and RIPK1 kinase-dependent cell death.

Fig. 3

a Representative images of the back skin and tail of 7-week old mice of the indicated genotype. The right panel shows H&E-stained skin sections of the respective mice. Scale bar: 200 μm. b Levels of IL-6, TNF and IL-17 in serum of 8-weeks-old OTULINfl/fl (WT; n = 9 or 11), ΔKerOTULIN (KO; n = 8), ΔKerOTULIN-TNFR1−/− (n = 7), ΔKerOTULIN TNFR1+/− (n = 7), ΔKerOTULIN-RIPK1D138N/D138N (n = 4), and ΔKerOTULIN/FADD/MLKL (n = 5) mice (Mann−Whitney two-sided test; *p < 0.5; **p < 0.01; ***p < 0.001; ****p < 0.0001). Data represent means ± SEM. c Epidermal thickness quantification at 7−11 weeks of age. NL non-lesional; L lesional (n = 10 per condition; ****p < 0.0001, One-way ANOVA with multiple comparisons). Data represent means ± SEM. d Primary keratinocyte cultures (n = 3 biological replicates per condition) were treated with 20 ng/ml mTNF with or without priming with IFN-γ (10 ng/ml) 8 h prior to TNF stimulation. Viability was assessed by Sytox Green uptake. Representative graphs for three independent experiments. (Residual maximum likelihood (REML); ***p = 0.002). Data represent means ± SEM. e Incucyte images depicting Sytox Green uptake by dead keratinocytes at 0 and 24 h post IFN-γ and TNF treatment. This experiment was repeated three times independently with similar results. f Western blot analysis on lysates from primary keratinocyte cultures isolated from OTULINfl/fl and ΔKerOTULIN mice that were treated with TNF for the indicated time-points. The representative figure for four independent experiments. g Ubiquitin pulldown by UBAN-IP on lysates from PMK cultures isolated from OTULINfl/fl and ΔKerOTULIN mice treated with TNF for the indicated time-points followed by immunoblotting for M1 Ubq chains. Pre-immunoprecipitation lysate (PIP) was immunoblotted for M1 Ubq (M1), RIPK1, and OTULIN. α-Actin is shown as loading control. This experiment was repeated three times independently with similar results.