Fig 2: Sensitivity of chemotherapeutic agents to CYLD-knockdown OSCC cells. Human OSCC cell line (SAS) cells were transfected with control siRNA (siN) or CYLD-specific siRNA (siCYLD) and then treated with cisplatin (A), 5-FU (B), carboplatin (C), docetaxel (D), and paclitaxel (E). The cell survival rates of SAS cells were assessed 72 h after treatment. Values are means ± S.D. of quadruple samples. **p < 0.01 vs siN group in Tukey–Kramer method

Fig 3: Effect of CYLD down-regulation on intracellular signaling pathways in OSCC cells. A Comprehensive changes in protein expression by CYLD knockdown were assessed by proteome analysis. B KEGG database analysis of cell signaling pathways activated by CYLD knockdown. C Phosphorylation of Akt and ERK was assessed by immunoblotting. Cells were transfected with the indicated siRNAs and then incubated for 72 h before harvesting. Cell lysate was immunoblotted with antibodies against the indicated proteins. D Cells were treated with LY294002 (Akt Inhibitor, 20 µM), PD98059 (ERK Inhibitor, 20 µM), combination, and cisplatin (8 µg/mL). Cells survival rate are assessed after 72 h. Values are means ± S.D. of triplicate samples. **p < 0.01 in Tukey–Kramer method

Fig 4: CaMKII-mediated phosphorylation upregulates CYLD deubiquitinase activity.Purified CYLD was pre-incubated with purified CaMKII in Ca2+/calmodulin-containing medium in the presence or absence of ATP. The samples were subsequently incubated with K63-linked polyubiquitins for different times, followed by Western immunoblotting with a pan-ubiquitin antibody. The rate of degradation of K63-linked polyubiquitins increased when CYLD was pre-incubated with CaMKII in ATP-containing medium. Parallel controls (Ctrl 60′) were heat-inactivated prior to incubation with K63-linked polyubiquitins for 60 minutes. Two experiments yielded similar results.

Fig 5: CaMKII co-immunoprecipitates with CYLD and promotes phosphorylation of CYLD at the PSD.(A) CYLD in solubilized PSD fractions was immunoprecipitated using CYLD antibody, followed by Western immunoblotting with antibodies for either CYLD or CaMKII. CaMKII co-immunoprecipitated with CYLD while neither protein was detected when beads without CYLD antibody were used. Two independent experiments yielded similar results. (B) PSD fractions were incubated under various conditions designed to manipulate CaMKII activity, followed by Western immunoblotting with CYLD antibody. Addition of Ca2+/calmodulin along with ATP caused a distinct mobility shift in CYLD (lane 3), indicative of phosphorylation. The mobility shift was prevented upon addition of CN21, a CaMKII inhibitor (lane 4). The control peptide had no appreciable effect on the observed mobility shift (lane 5). Two independent experiments yielded similar results. (C) PSD fractions were incubated under conditions designed to manipulate endogenous kinases and phosphatases at the PSD, followed by Western immunoblotting with CYLD antibody. The mobility shift elicited by ATP/Ca2+/CM was reversed upon incubation in the absence of the phosphatase inhibitor MicrocystinLR (lane 3).