Fig. 4.

Sharpin^cpdm/cpdm cells are more susceptible to CYLD-mediated cell death. (A) Sharpin^+/+, Sharpin^cpdm/cpdm, Cyld^−/−, and Sharpin^cpdm/cpdm Cyld^−/− ADF were treated with mTNF (10 ng/mL) for 24 h in the presence or absence of zVAD (20 μM) to induce necroptosis or cycloheximide (CHX, 1 μg/mL) to induce apoptosis. Cell death was analyzed by propidium iodide staining and flow cytometry. Data from three independent experiments are shown. Error bars represent mean ± SD, and one-way ANOVA analysis was performed. ***P < 0.001 Sharpin^cpdm/cpdm versus Sharpin^+/+ ADF. P value of Sharpin^cpdm/cpdm Cyld^−/− versus Sharpin^+/+ ADF is not significant. (B) Sharpin^+/+, Sharpin^cpdm/cpdm, Cyld^−/−, and Sharpin^cpdm/cpdm Cyld^−/− ADF were stimulated with mTNF (100 ng/mL) in the presence of CHX (1 μg/mL) for 3 and 6 h. Apoptosis was examined by blotting for cleaved CASP8 and cleaved CASP3 (C). For each panel, data shown are representative of at least three independent experiments. (C) Necroptosis was analyzed by blotting for phospho-MLKL (Ser-345) in Sharpin^+/+, Sharpin^cpdm/cpdm, Cyld^−/−, and Sharpin^cpdm/cpdm Cyld^−/− ADF stimulated with mTNF (25 ng/mL) in the presence of zVAD (20 μM) for 1.5 and 3 h. Result shown is representative of at least three independent experiments. (D) Sharpin^+/+, Sharpin^cpdm/cpdm, Cyld^−/−, and Sharpin^cpdm/cpdm Cyld^−/− ADF were stimulated with mTNF (100 ng/mL) in the presence of CHX (1 μg/mL) and zVAD (20 μM) for 2 and 4 h. The death-signaling Complex II was isolated by FADD immunoprecipitation and sequentially blotted for RIPK1 and FADD. Experiment was repeated twice with similar results. (E) Cyld^+/+ and Cyld^−/− MEFs transfected with small guide RNA (sgRNA) that is nontargeting (WT) or Sharpin-targeting (KO) were stimulated with mTNF (100 ng/mL) for 0, 5, and 15 min. Lysates were subjected to Flag-K63-TUBE pulldown to isolate K63-ubiquitinated proteins followed by blotting for RIPK1. Whole-cell lysates were also blotted for RIPK1 and ACTIN. Result shown is representative of three independent experiments. (F) Residues 411 to 445 of WT mouse CYLD encompassing the cluster of seven serines equivalent to those reported to be phosphorylated on human CYLD, and the corresponding ser-to-ala mutations in the nonphosphorylatable CYLD-S7A mutant are depicted. Ser-417 of mouse CYLD is equivalent to Ser-418 of human CYLD. Cyld^−/− MEF were transduced with retroviruses encoding a control (triangles), WT CYLD (circles), or CYLD-S7A (squares). Transduced cells plated in triplicates were treated with media or 0.1 ng/mL mTNF plus 20 μM zVAD-fmk and imaged for 24 h in the presence of YOYO-3. Aliquots of the complemented MEF were also analyzed by Western blotting to demonstrate defective phosphorylation in the mutant CYLD but equivalent expression of WT and mutant CYLD. Error bars represent mean ± SD from three biological replicates, and paired Student’s t test was performed. *P < 0.05.