Figure 4: IL-25 secreted by Q2-3-treated fibroblasts suppresses the growth of mammary tumour cells.

(a) Western blot analysis of the IL-25 secretion activity of mouse (3T3) and human (WI38) fibroblasts in response to Q2-3 treatment. Different fibroblast-conditioned media (CM), including 3T3-CM and WI38-CM, were collected from the 3D cultures and were stained with coomassie blue, revealing that the total protein level in tested CM was normalized. Aliquots of 3T3-CM and WI38-CM were immunodepleted for IL-25. Rabbit IgG (isotype control) was used as a negative control. Amounts of IL-25 (relative staining intensity) were further normalized with the value detected for Q2-3-treated 3T3-CM (blue) or Q2-3-treated WI38-CM samples (purple). (b) Reduction in cytotoxicity of 3T3-CM on 4T1 cells after immunodepletion of IL-25. The control (fresh) medium, 3T3-CM, Q2-3-treated 3T3-CM, Q2-3-treated 3T3-CM with added IL-25 protein, Q2-3-treated 3T3-CM with the immunodepletion of IL-25 and Q2-3-treated 3T3-CM with control IgG-mediated immunodepletion, were compared for their suppressive effect on growth of 4T1 cells. (c) Reduction in cytotoxicity of WI38-CM on MDA-MB-231 after immunodepletion of IL-25. Similarly, WI38-CM with added IL-25 protein, WI38-CM with the immunodepletion of IL-25, or Q2-3-treated WI38-CM with control IgG-mediated immunodepletion, were compared for their effect on growth of MDA-MB-231 cells. The growth activity of 4T1 cells or MDA-MB-231 cells was determined using MTT assay at 5 days post cultivation, and was normalized to the control group (with fresh medium only). Data are reported as mean±s.d. (n=3). *P<0.05; **P<0.01; ***P<0.001 (two-tailed Student’s t-test). (d) Western blot analysis of IL-17RA and IL-17RB (IL-25R) expression in MDA-MB-231 and MCF-10A cells. Knock down of human IL-25R (both 56 and 33 kDa molecules) expression in MDA-MB-231 cells was performed with three designed siRNAi treatments. Neg, negative control RNAi. (e) Western blot analysis of the cleavage of caspases 8 and 3 in MDA-MB-231 cells. MDA-MB-231 cells were treated by control-, Neg RNAi- or IL-25R RNAi, for 48 h. Some cells in each test group were cultivated with recombinant human IL-25 (200 ng ml⁻¹), WI38-CM, or WI38-CM with the immunodepletion of IL-25, for another 24 h. White arrowheads, cleaved protein. Black arrowheads, uncleaved protein. β-Actin served as an internal control. Similar results were obtained from three independent experiments.