Fig. 8
K63-linked ubiquitination of YAP induces its growth-promoting function. a Left panel: immunoblotting of YAP and GAPDH in HMLE cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). Right panel: growth curves. n = 5 biological replicates. b Images (upper panel) and quantification (lower panel) of soft agar colony formation by the cells described in (a). n = 3 biological replicates. c Endpoint images of tumors dissected from NSG mice with mammary fat pad injection of MDA-MB-231 cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). n = 10 (mock), 10 (YAP WT) and 9 (YAP 2KR) mice per group. d, e Tumor growth curves (d) and tumor weight (at the endpoint, e) of NSG mice with mammary fat pad injection of MDA-MB-231 cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). n = 10 (mock), 10 (YAP WT) and 9 (YAP 2KR) mice per group. f Left panel: immunoblotting of YAP, SKP2, and GAPDH in BT549 cells transduced with MYC-SKP2 with or without YAP shRNA. Right panel: growth curves. n = 5 biological replicates. g Left panel: immunoblotting of YAP, OTUD1 and GAPDH in BT549 cells transduced with OTUD1 shRNA with or without YAP shRNA. Right panel: growth curves. n = 5 biological replicates. h, i Kaplan–Meier curves of recurrence-free survival of patients with breast cancer, stratified by SKP2 (n = 3951 patients, probe: 203625_x_at, (h) or OTUD1 (n = 1764 patients, probe: 226140_s_at, (i) expression levels. Data were obtained from http://kmplot.com/analysis/. Auto-select best cutoff was used in the analysis. j Model for the regulation of YAP localization and activity by non-proteolytic ubiquitination. Error bars in a, b, and d–g are s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001
