Fig. 3
Transforming growth factor (TGF)-β increases interleukin (IL)-13 expression in the peripheral blood lymphocytes (PBLs) of patients with systemic sclerosis (SSc) via a Smad-dependent mechanism. a Type I TGF-β transmembrane receptor (TGF-βR1) and TGF-βR2 mRNA levels were measured by RT-PCR analysis in resting PBLs from healthy donors (n = 7) and patients with SSc (n = 11). b and c PBLs from patients with SSc (left panel) and healthy donors (middle panel) as Jurkat T cells (right panel) were cultured in 0.5 % fetal calf serum (FCS)-containing medium and treated with a specific Smad3 inhibitor (SIS3) for 1 h before addition of TGF-β or not for 4 h. b Expression of phosphorylated Smad3, nonphosphorylated Smad3 and β-actin was assessed by Western blotting. Semiquantitative analysis was performed by using ImageJ software, with β-actin levels used for normalization. Normalized data are schematized as bars under the Western blots. c Expression of IL-13 mRNA was measured by quantitative RT-PCR. The results are presented as mean percentage (±SD) of IL-13 expression detected in the presence of TGF-β (dark bars) relative to the respective IL-13 expression without TGF-β (white bars) and reported to 100 to allow comparison. d and e Jurkat T cells were transfected with d (CAGA)9-Lux or e pGL3 2666-bp IL-13 promoter construct/Luciferase (2666 bp-IL13-Lux) . Cells were then treated (dark bars) or not (white bars) with TGF-β in 0.5 % FCS-containing medium for 24 h. Jurkat T cells transfected with (CAGA)9-Lux d or 2666 bp-IL13-Lux promoter (e) were treated with or without SIS3 1 h before adding (or not) TGF-β (middle double bars). Jurkat T cells were cotransfected with c (CAGA)9-Lux promoter and a dominant negative of Smad3 (∆NSmad3) or e 2666 bp-IL13-Lux promoter and ∆NSmad3, and then Jurkat cells were treated with or without TGF-β (right double bars). The results are presented as mean percentage (±SD) of (CAGA)9-Lux or 2666 bp-IL13-Lux promoter activity detected in the presence of TGF-β (dark bars) relative to respective activity without TGF-β (white bars) and reported to 100 to allow comparison. b–e Results are representative of four independent experiments. **p < 0.01; ***p < 0.001