Figure 5

Extracellular ADAMTSL2 inhibits TGFβ signalling and myofibroblast differentiation in human cardiac fibroblasts. Human foetal cardiac fibroblasts (hfCFBs) treated with conditioned medium harvested from hfCFBs transduced with ADAMTSL2 (L2-medium) or vehicle control (Veh-medium, see Supplementary Fig. S3a). Data represent experiments from 3–5 different cell passages (n = 15). (a) Immunoblot of ADAMTSL2 in L2-medium and Veh-medium. (b) mRNA levels of transforming growth factor (TGF)β 1 (TGFB1), latent TGFβ binding protein 1 (LTBP1), connective tissue growth factor (CTGF), periostin (POSTN), and α-smooth muscle actin (α-SMA, encoded by ACTA2). (c) Representative immunoblots of phosphorylated (pSMAD2), total SMAD2/3 and α-SMA in whole-cell protein lysates and (d) quantification of blots. (e) Representative immunoblots and quantification of pSMAD2 and total SMAD2/3 in lysates from hfCFBs treated with Veh- or L2-medium, which was pre-incubated with active, TGFβ. hfCFBs were treated for 0 (T0), 30 (T30) or 60 (T60) minutes. Uncropped blots are available in Supplementary figure IV. Gene expression was normalized to RPL4. GAPDH was used as intracellular protein loading control. Data are presented as mean ± min/max values and statistical analysis was performed using the Student t-test vs. respective controls.