Fig. 3: LUBAC-mediated M1 poly-Ub controls MLKL-dependent production of inflammatory signaling molecules during necroptosis.
From: LUBAC-mediated M1 Ub regulates necroptosis by segregating the cellular distribution of active MLKL
A mRNA expression levels of CXCL1 and CXCL10 of control (UT), HOIPIN-8 (30 µM) or NSA (10 µM)-pre-treated HT-29 cells upon treatment with TBZ (10 ng/mL TNFα, 1 µM BV6, 20 µM zVAD.fmk) for 3 h. Gene expression was normalized against 18 S and RPII mRNA expression and is presented as x-fold mRNA expression compared to the untreated (UT) control. Mean and SEM of n = 4 independent experiments are shown. *P < 0.05; ***P < 0.001; ****P < 0.0001. B. mRNA expression levels of CXCL1 and CXCL10 of control (siCtrl) or HOIP knock-down (siHOIP) HT-29 cells treated with TBZ (10 ng/mL TNFα, 1 µM BV6, 20 µM zVAD.fmk) for 4 h. Gene expression was normalized against 18 S and RPII mRNA expression and is presented as x-fold mRNA expression compared to the untreated (UT) control cells. Mean and SEM of n = 4 independent experiments are shown. ****P < 0.0001. C. ELISA-based quantification of CXCL1 secretion in supernatants of control (UT) or HOIPIN-8 (30 µM)-pre-treated HT-29 cells after treatment with TBZ (10 ng/mL TNFα, 1 µM BV6, 20 µM zVAD.fmk) for the indicated time points. Mean and SEM of n = 3 independent experiments are shown. **P < 0.01; ***P < 0.001. D. ELISA-based quantification of CXCL1 secretion in supernatants of control (siCtrl) or HOIP knock-down (siHOIP) HT-29 cells treated with TBZ (10 ng/mL TNFα, 1 µM BV6, 20 µM zVAD.fmk) for the indicated time points. Mean and SEM of n = 4 independent experiments are shown. **P < 0.01; ***P < 0.001. E. MACE-seq-determined heatmap showing the row-wise scaled intensity (left panel) of the top-25 upregulated genes in HT-29 cells treated with TBZ (10 ng/mL TNFα, 1 µM BV6, 20 µM zVAD.fmk) for 3 h in comparison to HOIPIN-8 (30 µM)-pre-treated and TBZ-treated HT-29 cells. Genes are sorted according to their log2 fold change values (right panel). *P < 0.05. See also Figure S3. Statistical significance was determined using 2-way ANOVA followed by Tukey’s multiple comparisons test (A–D).
