Fig. 4

ATX inhibition of human VICs isolated from FCAVD patients causes the downregulation of the TGF-β pathway and related fibrosis processes. A The protein levels of p-Smad2, p-Smad3, Smad2/3, and GAPDH in VICs treated with TGF-β (5 ng/mL) for 0, 5, 10, 30, or 60 min in the presence or absence of BBT-877 (1 μM). B Quantification of phospho-Smad2 levels relative to the total Smad2/3 concentration. Immunoblots were quantified using ImageJ software. C, D Protein levels of p-p38, p38, p-AKT, AKT, p-ERK, ERK, p-JNK, and JNK in VICs treated with TGF-β (5 ng/mL) for 0, 5, 10, 30, or 60 min in the presence or absence of BBT-877 (1 μM). E, F mRNA expression levels of TGF-β receptor 1 (TGFBR1), TGF-β receptor 2 (TGFBR2), and cellular communication network factor 2 (CTGF, CCN2) in VICs after 24 h of TGF-β (5 ng/mL) stimulation in the presence or absence of BBT-877 (1 μM). G CTGF in VICs-conditioned media after 24 h of TGF-β (5 ng/mL) stimulation in the presence or absence of BBT-877 (1 μM). Data are presented as the mean ± SD and the experiments were performed independently in triplicate. **P < 0.01, ***P < 0.001 versus the vehicle control. P values were obtained using a two-tailed t-test