Fig 2: 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.

Fig 3: Aberrant EHMT2 and H3K9me2 expression. Representative western blot analysis of (A) EHMT2 protein expression (relative to ß-actin) and (B) validation of H3K9me2 level of mCyld+/+ and mCyld-/- cell lines (relative to H3). Western blot analysis of (C) EHMT2 protein expression (relative to ß-actin) and (D) quantification of H3K9me2 level of hCYLD+ and hCYLD- cell lines (relative to H3). *P<0.05. EHMT2, euchromatic histone lysine methyltransferase 2; H3, histone 3; m, mouse; h, human; CYLD, CYLD lysine 63 deubiquitinase.

Fig 4: MiR-381-3p does not directly downregulate the known common molecules involved in TNF-induced NF-κB signaling and cell death. (A) HT-29 cells were transfected with miR-NC or miR-381-3p for 60 h and then treated with the control PBS or TNF-α for 10 min, phos-IκB-α protein level was detected by western blot. (B) HT-29 cells were transfected with miR-NC or miR-381-3p for 60 h, and mRNA levels of Tnfr1, Tradd, Traf2, and Ripk1 were determined via qRT-PCR. (C) HT-29 cells were transfected with miR-NC or miR-381-3p for 60 h. Cell lysates were collected and subjected to western blot analysis of TNFR1, TRADD, TRAF2, and RIPK1. (D) MiR-381-3p and its putative binding sequence in the 3′-UTR of CYLD. (E) MiR-NC or miR-381-3p was co-transfected with pmir-GlO-CYLD-3′UTR into HEK-293T cells, and luciferase activity was measured. (F) HT-29 cells were transfected with miR-381-3p for 60 h, and then were treated with the control PBS, TNF-α/Smac mimetic or TNFα/Smac mimetic/z-VAD for 6 h. Cell lysates were collected and subjected to western blot analysis of CYLD protein level. **P < 0.01.

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.