Fig. 2

The effect of TGFβ on IL-33 expression by fibroblasts over time. Non-IPF (n = 5) and IPF (n = 5) HLFs were stimulated with 2 ng/ml of TGFβ for 4, 8 and 24 h and IL33 gene expression measured by qPCR. For each donor, all data was normalised to the unstimulated media alone control at 4 h (A). Non-IPF (n = 5) and IPF (n = 4) HLFs were stimulated with 2 ng/ml of TGFβ for 8 and 24 h, total protein isolated and 20 μg protein/lane separated by SDS-PAGE. IL-33 protein expression by non-IPF (B) and IPF (C) HLFs was assessed by western blot and quantified relative to α-Tubulin via densitometry (D). Representative cropped western blots from non-IPF and IPF donors are shown. Whole IL-33 blots for the representative non-IPF and IPF donors are viewable in Additional file 1: Figs. S2 and Fig. S3 respectively. IL-33 release from non-IPF (n = 3) and IPF (n = 3) HLFs treated with 2 ng /ml TGFβ for 4, 8 or 24 h was measured by ELISA. Recombinant IL-33 was titrated as a positive control (E) and analysed alongside neat HLF supernatants (F) Non-IPF and IPF donors shown in black and grey respectively. Bars indicate median values and each data point represents a single donor with statistical analysis performed by Wilcoxon signed-rank test