Figure 3: Q2-3 treatment significantly upregulates IL-25 expression in tumour-associated fibroblasts, as evaluated by 3D cell co-culture system.

(a) Effect of Q2-3 on population change of FSP-1⁺ER-TR7⁺ cells and their IL-25 expression level in lungs of test mice, quantified using flow cytometry. The percentage of IL-25⁺ fibroblasts in gated FSP-1⁺ER-TR7⁺ cells were compared with corresponding IL-25⁺ fibroblast population in PBS group. Data are reported as mean±s.d. (n=3). *P<0.05; ***P<0.001; NS, not significant (two-tailed t-test). FSP-1, fibroblast specific protein-1; ER-TR7, Erasmus University Rotterdam-thymic reticulum-7.IF staining; lung tissues of control (0.1% DMSO in PBS), Q2-3-treated and docetaxel-treated mice were collected at 21 days post tumour resection and stained for the presence of IL-25-expressing cells (red and indicated with arrowheads), FSP-1 (green) and DAPI (blue). T, tumour; A, alveoli. (b) IF staining, lung tissues of control (0.1% DMSO in PBS), Q2-3-treated and docetaxel-treated mice were collected at 21 days post tumour resection and stained for the presence of IL-25-expressing cells (red and indicated with arrowheads), FSP-1 (green) and DAPI (blue). T, tumour; A, alveoli. (c) Construction of 3D cell co-culture system for mammary tumour cells and tumour-associated fibroblasts. Western blot analyses of the expression of (d) mouse IL-25 in 3T3 fibroblasts and (e) human IL-25 in WI38 fibroblasts, in response to Q2-3 treatments in 3D culture. Some fibroblasts in the upper layer were co-cultured with mammary tumour cells as indicated. The expression level of IL-25 in 3T3 and WI38 fibroblasts were quantified and normalized using ImageJ software. Fold changes of IL-25 expression level in test samples were normalized with the value in fibroblasts of the 0 h group and were indicated by the number labelled in blue. β-Actin served as an internal control. Similar results were obtained from three independent experiments.