Fig 2: CYLD inhibits EBV reactivation dependent on p18.a Representative IHC staining of CYLD and EAD expression taken from pathological sections of NPC patients (100×: scale bar, 100 µm; 400×: scale bar, 50 µm). b EAD expression level was calculated based on CYLD expression. High and low groups were according to median expression. c, d HK1-EBV and HONE1-EBV cells infected with vector or CYLD and total RNA from cells was isolated and subjected to real-time PCR. e, f Real-time PCR showing EBV DNA copy levels of EBV in HK1-EBV and HONE1-EBV cells. HONE1-EBV cells were infected with p18 lentiviral shRNAs and then transfected with the indicated constructs. g Cell lysates were then extracted and subjected to Western blotting. h Total RNA and i gDNA were isolated and subjected to real-time PCR. j Cell lysates were immunoblotted with antibodies against the indicated proteins. ß-Actin was used as a control to confirm equal loading of protein. (Data are represented as mean ± SEM (n = 3). Differences were considered significant at *p < 0.05, **p < 0.01, ***p < 0.001).

Fig 3: Overexpression of FLAP correlates to CYLD suppression in resection tissues of HCC patients. (A) Representative immunohistochemistry (IHC) images of FLAP, MGST2, CysLTR2, and CYLD in each subject using the indicated antibodies. Scale bar = 20 µm, bar = 10 µm (magnification). (B) The expression levels of FLAP, mGST2, CysLTR2, and CYLD were clustered. Patient and donor codes are indicated. Grades of the indicated markers (Red: ++ = brown, Blue: + = golden, Green: ND = not detected). (C) The expression levels of CD34 localization and positive rate (%) for Ki-67 were clustered. Patient and donor codes are matched with each other. For each analysis, n = 10/per group.

Fig 4: Effect of CYLD on RAD18, DTL, POLH and PCNA monoubiquitination. (A) MCF-7 cells were transfected with CYLD-targeted siRNA (siCYLD) or siControl. At 65 h post-transfection, the cells were irradiated at 20 J/m2 UV and harvested 1 or 6 h post-irradiation. (B) MCF-7 cells were transfected with siRNA against CYLD, RAD18 and DTL or their double or triple combinations. At 65 h post-transfection, the cells were UV-irradiated at 20 J/m2 and harvested 6 h post-irradiation. Whole-cell extracts were fractionated by SDS-PAGE and analyzed by Western blot with indicated antibodies. Protein levels were normalized to GAPDH and presented each relative to its level in unirradiated or UV-irradiated siControl-treated cells.

Fig 5: OTULIN and MALT1 simultaneously bind to the PUB domain of HOIP. (A) WT and various mutants of Myc-HOIP were co-expressed with FLAG-MALT1 and/or FLAG-OTULIN in HEK293T cells, as indicated. Cell lysates and anti-Myc immunoprecipitates were immunoblotted with the indicated antibodies. (B) The endogenous association of CYLD, OTULIN, LUBAC, and the CBM complex. Jurkat cell lysates were immunoprecipitated with normal mouse IgG or anti-MALT1 antibody, and subjected to immunoblotting by the depicted antibodies. *; nonspecific signal. (C) Schematic illustration of the LUBAC/DUBs-CBM complex interaction.