Fig. 3

Gene and protein expression of myofibroblast phenotype markers and ECM macromolecules in nintedanib-treated SSc fibrocytes. A Quantitative real time polymerase chain reaction (qRT-PCR) of the gene expression of αSMA, S100A4, COL1, and FN in cultures of fibrocytes isolated from SSc patients maintained in normal growth medium without any treatment and treated with nintedanib at the concentrations of 0.1 μM and 1 μM for 3 h. The qRT-PCR is performed on 16 independent in vitro experiments derived from fibrocytes isolated from each enrolled SSc patient. Gene expression corresponds to the expression level (fold-increase) of the target gene of nintedanib-treated SSc fibrocytes compared with that of untreated cells, taken as the unit value by definition [18]. B Western blotting and related densitometric analysis of protein synthesis of αSMA, S100A4, COL1, FN, and GAPDH in SSc fibrocytes maintained in normal growth medium without any treatment and treated with nintedanib at the concentrations of 0.1 μM and 1 μM for 3 and 24 h. Western blotting is performed on 10 independent in vitro experiments with fibrocytes isolated from 6 SSc patients. For each experimental condition, the value for the synthesis of αSMA, S100A4, COL1, and FN is normalized to that of the corresponding GAPDH. The resulting value of each treatment is compared with that of the related untreated cells (taken as unit value). Data of qRT-PCR and Western blotting are expressed as median with range