Fig. 4

Systemic sclerosis (SSc) mesenchymal stem cells (MSCs) silenced by small interfering RNA (siRNA) CD248. a qRT-PCR of CD248 in ten SSc-MSCs (five early-onset subset [EOS] and five long-standing subset [LSS]) transfected with specific CD248-siRNA or scrambled (scr)-siRNA. Cells transfected with CD248-siRNA show a decreased expression of the CD248 gene compared with cells transfected with scr-siRNA. In SSc-MSCs treated with scr-siRNA, the transforming growth factor (TGF)-β stimulus induces a significant decrease of CD248 expression. On the contrary, in CD248-siRNA cells, TGF-β is unable to modulate CD248 mRNA expression. b qRT-PCR of α-SMA in ten SSc-MSCs (five EOS and five LSS) transfected with specific CD248-siRNA or scr-siRNA. In SSc-MSCs treated with scr-siRNA, the TGF-β stimulus induces a significant increase of α-SMA mRNA expression. On the contrary, in CD248-siRNA cells, TGF-β is unable to modulate α-SMA mRNA expression. c qRT-PCR of Ki-67 in ten SSc-MSCs (five EOS and five LSS) transfected with specific CD248-siRNA or scr-siRNA. In SSc-MSCs treated with scr-siRNA, the PDGF-BB stimulus induces a significant increase of Ki-67 mRNA expression. On the contrary, in CD248-siRNA cells, PDGF-BB is unable to modulate Ki-67 mRNA expression. The TGF-β isoform used is TGF-β1. Any single dot in the figure represents the median of triplicate experiments for each patient. *** p = 0.0001. d Western blot analyses performed in four SSc-MSCs (two EOS and two LSS) confirmed the results observed by qRT-PCR analyses. Pictures are representative of all experiments. e and f Densitometric analysis of (e) CD248 protein bands and (f) α-SMA protein bands. The values were expressed as protein relative quantification/β-actin relative quantification. * p = 0.02