Fig. 3

SB-505124 fully and (5Z)-7-Oxozeaenol partly inhibits transforming growth factor β1 (TGFβ1)-induced Smad-dependent gene expression whereas LDN-193189 does not. a Primary bovine chondrocytes were pre-incubated with 5 μM SB-505124, 0.5 μM (5Z)-7-Oxozeaenol or 0.05 μM LDN-193189 for 1 h, and subsequently stimulated with 1 ng/ml TGFβ1 for 2 h and 24 h. With the use of quantitative (q)PCR, expression of bSerpine1 was measured as response gene for pSmad3 signaling after 2 h, and bId1 for pSmad1/5 signaling after 2 h. b The pSmad3 responsive CAGA₁₂-luciferase (CAGA ₁₂ -luc) construct was placed in primary chondrocytes with the use of an adenovirus, and cells were stimulated with 1 ng/ml TGFβ1 for 8 h with or without inhibitors after 2 days. c Primary chondrocytes were stimulated with 1 ng/ml TGFβ1 for 24 h and gene expression was measured using validated cDNA-specific primers and qPCR. MMP matrix metalloproteinase, NGF nerve growth factor, ALK5 activin receptor-like kinase 5. d Gene expression was measured after pre-incubation of chondrocytes for 1 h with inhibitors followed by stimulation with 1 ng/ml TGFβ1 for 24 h. For qPCR data, average ± sd (mean ± sd) was plotted, with each dot representing the average of one donor (a) or four donors (c and d). Analysis was performed using one-way analysis of variance with Tukey’s post-hoc test: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. A change of 1 ΔΔC _t equals twofold upregulation