Figure 5.

IL11 alone is not sufficient to trigger fibroblast activation in vitro in comparison to TGFβ. (A). Selected fluorescence images of αSMA and Collagen 1A1 immunostaining in primary human lung fibroblasts treated with indicated cytokines (TGFβ, 10 ng/ml; IL11, 10 ng/ml; All cytokine treatment lasted for 24 h; scale bar 100 µm; magnification 20 x); αSMA ROI was quantified using cytoskeletal rearrangement assay tool from the HCS Studio software. Collagen 1A1 ROI was quantified using the compartmental analysis from the HCS Studio software. Details regarding ROI quantification are included in the methods section. (B). Quantification of fluorescence intensity of αSMA and Collagen 1A1 immunostaining in primary human lung fibroblasts treated with indicated cytokines (TGFβ, 10 ng/ml; IL11, 10 ng/ml; All cytokine treatment lasted for 24 h). Representative of 2 independent experiments, mean and SD of 3 technical replicates are depicted. (C). Quantification of intracellular αSMA protein expression by HTRF in primary human lung fibroblasts treated with indicated cytokines (TGFβ, 10 ng/ml; IL11 low, 10 ng/ml; IL11 high, 100 ng/ml; All cytokine treatment lasted for 72 h). (D). Quantification of secreted pro-fibrotic and pro-inflammatory mediators (TIMP1, left; CTGF, middle; IL6, right) from primary human lung fibroblasts treated with indicated cytokines by ELISA (TGFβ, 10 ng/ml; IL11 low, 10 ng/ml; IL11 high, 100 ng/ml; All cytokine treatment lasted for 72 h). Representative of at least 2 independent experiments. P-values were determined by one-way ANOVA or Student’s t-tests. **P ≤ 0.01; **** P ≤ 0.0001; ns, not significant. Bar graphs represent means and standard deviations of two independent experiments.