Fig. 4

Implication of the p38 mitogen-activated protein kinase pathway downstream of transforming growth factor (TGF)-β receptors on peripheral blood lymphocytes (PBLs). PBLs from a patients with SSc and b healthy donors were treated with (or without) SB431542 (activin receptor-like kinase ALK5 inhibitor) or SB203580 (specific p38 inhibitor) for 1 h before the adding, or not, TGF-β. Interleukin (IL)-13 mRNA expression was measured by RT-PCR. The results are presented as mean percentage (±SD) of IL-13 expression detected in the presence (dark bars) or absence (white bars) of TGF-β relative to basal IL-13 expression taken as 100. c After 16 h of culture in 0.5 % fetal calf serum (FCS)-containing medium, Jurkat T cells were treated with (or without) SB431542 or SB203580 for 1 h before adding, or not, TGF-β, and IL-13 mRNA expression was measured by RT-PCR (left panel). Jurkat T cells were transfected with pGL3 2666-bp IL-13 promoter construct/Luciferase (2666 bp-IL13-Lux) or empty pGL3-Lux promoter and then treated or not with SB431542 or SB203580 for 1 h before adding TGF-β (dark bars) or not (white bars) for a further 24-h culture in 0.5 % FCS-containing medium (right panel). The results are presented as mean percentage (±SD) of IL-13 expression (left panel) or 2666 bp-IL13-Lux promoter activity (right panel) detected in the presence (dark bars) or absence (white bars) of TGF-β relative to basal IL-13 expression taken as 100. The results shown in a–c are representative of four independent experiments. *p < 0.05; ***p < 0.001. d Phosphorylated Smad3 and respective nonphosphorylated protein expression was detected by Western blotting in PBLs from patients with SSc (left panel), healthy donors (middle panel) and Jurkat T cells (right panel) cultured in 0.5 % FCS-containing medium with SB431542 or SB203580 for 1 h before addition of TGF-β or not for 4 h. The results are representative of four independent experiments. Semiquantitative analysis of blots was performed by using ImageJ software with Smad3 levels for normalization. Normalized data are schematized as bars under the Western blots