Fig. 4.

Inflammatory cytokines trigger Hippo signaling activation and YAP degradation. a Gene expression analysis of Ctgf and Cyr61 in primary chondrocytes after treatment with TNFα or IL-1β for 24 h, respectively. b Luciferase reporter assay of Gal4/TEAD4-reporter in primary chondrocytes treated with different doses of TNFα for 24 h. c Luciferase analysis of Gal4/TEAD4-reporter activity in HEK293A cells transfected with Flag-tagged YAP for 24 h and subjected to TNFα treatment for 6 h. d Phos-tag gel and immunoblot analyses of Hippo components in primary articular chondrocytes isolated from newborn mice after treatment with TNFα or IL-1β at 5 ng/ml, respectively. Arrows indicate the phosphorylated form of proteins. e Western blot analysis of YAP and TAZ in primary articular chondrocytes treated with TNFα or IL-1β at 5 ng/ml respectively for the indicated time. f Western blot analysis of YAP and TAZ expression in HEK293A cells with pre-treatment of CHX for 2 h followed by treatment with TNFα for 6 h together with MG132 or Chlq (chloroquine). g Primary chondrocytes were transfected with indicated plasmids and harvested for ubiquitination analysis 6 h after treatment with MG132 at 10 μM and TNFα at 5 ng/ml. Arrows indicate the degraded YAP. h Ubiquitination analysis of YAP after TNFα treatment at 5 ng/ml for the indicated time in HEK293T cells. i Immunofluorescence staining of YAP in primary chondrocytes after treatment with TNFα at 5 ng/ml. Scale bars, 20 μm. All data are presented as mean ± SD. *p < 0.05, ^**p < 0.01, ^***p < 0.001. a, b One-way ANOVA followed by Dunnett’s test was performed with 0 ng/ml group as control. c One-way ANOVA followed by Tukey’s test was used. All experiments were repeated three times independently