Fig 2: Premature thymic involution and over-activation of NF-κB in the thymus of K5-CYLDC/S mice. (A-C) Analysis of the expression of the transgene by immunostaining with a specific antibody against the HA tag. Expression of HA is detected in the medulla of the thymus of the K5-CYLDC/S mice (B), following the expression pattern of the K5 (C), while it is not detected in the Controls (A). (D) Analysis by WB of the expression of the transgene in protein extracts from isolated thymic cells of mice of 3.5-month-old. Note the overactivation of NF-κB (increased levels of P-p65) in the K5-CYLDC/S mice. Mann-Whitney U test was used for statistical analysis. (*p<0.05). (E, F) Histological analysis of the thymus of 2.5-month-old mice. Observe the expansion of the cortical zone and reduction of the medullar region in the thymus of transgenic mice (F). (G, H) H&E staining of 3.5-month-old Control (G) and K5-CYLDC/S mice (H) thymus. A representative image of the thymic atrophy and infiltration of white adipose tissue in the thymus of transgenic mice (H) is shown. (I) Western blot showing the decreased expression of CYLD with age in the thymus of control mice. M, medulla. C, cortex. a, adipose tissue. Scale bars: 200 μm (A, B); 300 μm (C); 350 μm (E-H).

Fig 3: Histological and molecular signs of premature aging in the back skin of transgenic mice. (A-H) Representative histological images showing the back skin of 20-month-old Control mice (A) and the severe aging phenotype of the back skin of 20-month-old transgenic mice (B-D). (B-D) Note severe epidermal atrophy (compare insets in A with those of B and C; double-headed red arrow in D); foci of papillomatous hyperplasia (red arrows in C and D); numerous hyperplastic sebaceous glands, most of them orphan and grouped in the dermis (D); reduced number of HFs, and scarce or even lack of adipose tissue (compare A with B-D) in the back skin of the K5-CYLDC/S mice. (E-H) Tail skin of Control (E) and transgenic (F-H) mice. Note the presence of hyperplastic sebaceous glands and extensive epidermal atrophy (compare inset in E with those in F and G) in the tail of the K5-CYLDC/S mice. (I, J) WB of total protein extracts from skin of 12-month-old (I) and 6-month-old (J) showing elevated levels of p16, p19 and γH2AX in the K5-CYLDC/S mice. Tubulin and Actin are used as control loading. (K) WB of total protein extracts from the skin of Control mice from 1 to 33 months of age showing the decreased expression of CYLD as mice age. GAPDH is used as a control loading. White arrows: sebaceous glands; red arrows: papillomatous hyperplasia; double-headed red arrows: areas of epidermal atrophy. Scale bars: 250 μm (A-D); 200 μm (E-H).

Fig 4: Only cFLIPL is dependent on LUBAC to induce NF-?B, whereas all cFLIP isoforms require TAK1. A, HEK293 cells were transduced with lentivectors encoding either scrambled control shRNA (scr) or shRNAs targeting HOIL-1, HOIP, and SHARPIN sequentially. Generation of LUBAC knockdown (KD) cells was confirmed by immunoblotting against each component of the complex and the housekeeping protein GAPDH. ns, nonspecific. B, TNF-a-induced NF-?B activation which was measured 6 h after stimulation at 10 ng/ml concentration is inhibited in LUBAC knockdown cells. C, scrambled and LUBAC knockdown HEK293 cells were co-transfected with an NF-?B firefly luciferase reporter construct (300 ng/well) and a Renilla luciferase reporter vector (normalization control, 100 ng/well) with empty or transactivator-expressing pcDNA3 vectors (500 ng/well). The luciferase reporter assay was performed 24 h post-transfection. D, immunoblot showing the expression of ectopically expressed CYLD in HEK293 cells. Cells transduced with a GFP-expressing lentivector were used as controls. Effects of CYLD overexpression on NF-?B activation levels induced by TNF-a (E) or cFLIPs, vFLIP, and Tax (F) were measured as described above. G, shRNA-mediated stable silencing of TAK1 and MEKK3 in HEK293T cells was validated by immunoblotting. H, these cells were further transduced with NF-?B luciferase lentivector to develop NF-?B sensor cell lines. Luciferase reporter assays were then performed 6 h following stimulation with TNF-a (10 ng/ml) or 48 h following transduction with control (enhanced GFP) or test lentivector (I). Results shown are mean ± S.D. (error bars) of a representative experiment of three, performed in triplicates. The two-tailed Student's t test was used to determine the statistical differences between two groups. RLU, relative luciferase units; LLV, luciferase lentivector.