Fig 2: 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 β-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 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 JIB04 on Cyld+/+ and Cyld−/− melanoma cells. Representative (A) western blot of H3K9me2 levels (relative to H3) following 24 h treatment with JIB04 (500 nM) and (B) anchorage-dependent clonogenic assay in murine cell lines. Representative (C) western blot of H3K9me2 levels (relative to H3) following 24 h treatment with JIB04 (500 nM) and (D) anchorage-dependent clonogenic assays of h cell lines (Cyld+/+:Cyld−/− colony size). JIB04, Jumonji histone demethylase inhibitor; Ctrl, control; h, human; m, mouse; CYLD, CYLD lysine 63 deubiquitinase; H3, histone 3.

Fig 5: Effect of histone inhibitor Chaetocin on Cyld+/+ and Cyld−/− melanoma cells. Representative (A) western blot of H3K9me2 and H3K9me3 levels following 24 h treatment with Chaetocin (10 nM) relative to H3 and (B) anchorage-dependent clonogenic assay of murine cell lines. Representative (C) western blot of H3K9me2 and H3K9me3 levels following 24 h treatment with Chaetocin (10 nM) relative to H3 and (D) anchorage-dependent clonogenic assay (hCyld+/+:hCyld−/− cell colony size). *P<0.05. CYLD, CYLD lysine 63 deubiquitinase; H3, histone 3; ctrl, control; m, mouse; h, human.