Fig. 4.

In cartilage, SB-505124 fully inhibits and (5Z)-7-Oxozeaenol partly inhibits transforming growth factor β1 (TGFβ1)-induced Smad2/3 phosphorylation and gene expression, whereas LDN-193189 does not. a Bovine cartilage explants were pre-incubated ex vivo 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, after which phosphorylated Smads were visualized by western blot using specific antibodies. kDa kilodalton. b With the use of quantitative (q)PCR, expression of bSerpine1 was measured as response gene for pSmad3 signaling after 24 h, and bId1 for pSmad1/5 signaling after 24 h. c Gene expression was measured after pre-incubation of explants 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 (b) or four donors (c). 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 or downregulation. TGF transforming growth factor, COL2A1 = collagen type II alpha 1 chain MMP matrix metalloproteinase, NGF nerve growth factor, ALK5 activin receptor-like kinase, JUNB = JunB proto-oncogene, FN1 = fibronectin 1