Figure 7.

Effect of signal transducer and activator of transcription 6 (STAT6) on IL-4 secretion in vitro. (A) Concentration of IL-4 (pg/ml) from bronchoalveolar lavage fluid in tumor-bearing wild-type (WT) and STAT6^−/− mice. (B) Concentration of IL-4 (pg/ml) in the serum when CD11b⁺ cells were cocultured with lung cancer cells (LLC1) using Transwell plates. CD11b⁺ cells were obtained for bone marrow of mice. (C) Secretion of IL-4 was measured by ELISA in the cocutured medium of tumor cells [STAT6^+/+ or STAT6^−/− with CD11b⁺ cells (STAT6^+/+ or STAT6^−/−)]. CON-Tumor: LLC1 tumor cells. Si-Tumor: STAT6 knockdown in LLC1 cells. CON-CD11b: CD11b⁺ cells. Si-CD11b: CD11b⁺ cells from STAT6^−/− mice. CON-CD11b^WTco: cocultured medium of LLC1 cells with STAT6^+/+CD11b⁺ cells for analysis of IL-4 expression. CON-CD11b^STAT6co: cocultured medium of LLC1 cells with STAT6^−/−CD11b⁺ cells for analysis of IL-4 expression. Si-CD11b^WTco: cocultured medium of STAT6^−/−LLC1 cells with STAT6^+/+CD11b⁺ cells for analysis of IL-4 expression. Si-CD11b^STAT6co: cocultured medium of STAT6^−/−LLC1 cells with STAT6^−/−CD11b⁺ cells for analysis of IL-4 expression. (D) mRNA expression of IL-4 was analyzed using RT-PCR. CD11b⁺ cells were isolated from STAT6^−/− mice and WT littermates. CON-CD11b: CD11b⁺ cells from bone marrow of WT mice. Si-CD11b: CD11b⁺ cells from STAT6^−/− mice. CON-Tumor: LLC1 tumor cells. Si-Tumor: STAT6 knockout in LLC1 cells. CON-Tumor^WTco: LLC1 cells were separated after 48 h of coculture with CD11b⁺ cells. CON-Tumor^STAT6co: LLC1 cells were separated after 48 h of coculture with STAT6^−/−CD11b⁺ cells for analysis of IL-4 expression. Si-Tumor^WTco: STAT6^−/−LLC1 cells were separated after 48 h cocultured with STAT6^+/+CD11b⁺ cells for analysis. Si-Tumor^STAT6co: STAT6^−/−LLC1 cells were separated after 48 h of coculture with STAT6^−/−CD11b⁺ cells for analysis. CON-CD11b^WTco: STAT6^+/+CD11b⁺ cells were separated after 48 h of coculture with STAT6^+/+LLC1 cells. CON-CD11b^STAT6co: STAT6^+/+CD11b⁺ cells were separated after 48 h of coculture with STAT6^−/−LLC1 cells. Si-CD11b^WTco: STAT6^−/−CD11b⁺ cells were separated after 48 h of coculture with STAT6^+/+LLC1 cells. Si-CD11b^STAT6co: STAT6^−/−CD11b⁺ cells were separated after 48 h of coculture with STAT6^−/−LLC1 cells for analysis of IL-4 expression. (E–H) CCK8 proliferation analysis of human lung cancer-derived cells (A549, H1299, SPC-A1) and mouse lung cancer-derived cells (LLC1) after genetic knockdown of STAT6. The knockdown efficiencies of si-STAT6 in A549, H1299, SPC-A1, and LLC1 were 85, 90, 97.5, and 88%, respectively, as verified by RT-PCR. (I,J) Representative results of cell cycle analysis in WT and si-STAT6 groups. (K,L) Comparative percentages of cells in G1, S, and G2/M between WT and STAT6^−/− groups in A549 and LLC1 cells. (M) Flow cytometry analysis of apoptosis. (N) Percentages of apoptotic cells in the WT and STAT6^−/− groups (annexin-V-positive, PI-positive or -negative). (O) Percentages of early apoptotic cells (annexin-V-positive, PI-negative). si-STAT6, STAT6 knockdown.