Fig 2: PGG selectively inhibits phosphorylation of TAK1 at the Thr184/187 site to inhibit its kinase activity. (A) RASF was pretreated with PGG (1-5 µM) for 2 hr, followed by IL-1ß stimulation for 30 minutes. Cell lysates were analyzed for MYD88, IRAK-1, IRAK-M, CYLD, A20, TAB1, pTAK1 (Thr184/187), TAK1, TRAF6, I-kBa, p38, pJNK, p-c-Jun, and ß-actin. (B) PBMC were pretreated with PGG (5 µM), followed by stimulation with a TLR2 agonist (PamCys3, 1 µg/ml), or TLR4 agonist (lipopolysaccharide, LPS; 1 µg/ml), IL-6 and IL-8 IL-6 and IL-8 production was determined in the conditioned media using commercially available ELISA kits. The results in each figure represent the experiments repeated on four RASF from different donors. (C) RASFs pretreated with PGG (10 µM) and stimulated with IL-1ß, immunoprecipitated (IP) with O-GlcNAc or TAB1, and probe for TAK1, TAB1, and p65, also included input for O-GlcNAc to show equal loading. #p<0.05 IL-1b vs. IL-1b+PGG; **p<0.01 for IL-1ß vs IL-1ß+ PGG.

Fig 3: Somatic mutation analysis. Bar plots showing the number of nucleotide variants found in both mCyld−/− cell lines but not in mCyld+/+ cell lines. Each bar represents the number of variants per 10 million base pairs along the chromosomal location in mCyld−/− cell line #1 (red) and #2 (blue). Common variants found in both mCyld−/− cell lines are only present on Chr 8 (gray) due to knockout of Cyld. Chr, chromosome; CYLD, CYLD lysine 63 deubiquitinase.

Fig 4: Effect of histone inhibitor CM272 on Cyld+/+ and Cyld-/- melanoma cells. Representative western blot of H3K9me2 levels following 24 h treatment with CM272 relative to H3 of (A) murine cell lines. Representative images of anchorage-dependent clonogenic assays of (B and C) murine cell lines (mCyld+/+:mCyld-/- colony size). Representative western blot of H3K9me2 levels following 24 h treatment with CM272 relative to H3 of (D) human cell lines and representative images of anchorage-dependent clonogenic assays of (E and F) human cell lines (hCYLD+:hCYLD- colony size). Representative western blot of CYLD protein expression following 24 h treatment with CM272 relative to ß-actin of (G) murine and (H) human cells. (I) Analysis of the chromatin accessibility of the mCyld-/- cell lines after 24 h treatment with CM272 (200 nM) (quantification of relative chromatin accessibility to untreated mCyld-/- cells). *P<0.05. Ctrl, control; h, human; m, mouse; CYLD, CYLD lysine 63 deubiquitinase; H3, histone 3.

Fig 5: Model of CYLD deficiency in melanoma cells. The upregulation of EHMT2 and associated di-methylation of H3K9 leads to heterochromatin and the induction of proliferation of proliferation. CM272, which inhibits the activity of EHMT2. The associated decrease in H3K9me2 levels results in loosening of chromatin and decreased proliferation of CYLD-deficient melanoma cells (abstracted and modified from smart.servier.com/). EHMT2, euchromatic histone lysine methyltransferase 2; CYLD, CYLD lysine 63 deubiquitinase.