Research Paper Volume 12, Issue 24 pp 25845—25864

Figure 2. Effects of FGF9 on bladder cancer cell aggressiveness and macrophage M2 polarization. (A) FGF9 knockdown was generated in the T24 bladder cancer cell line by transfection of si-FGF9. The transfection efficiency was validated by real-time PCR. P<0.01, student’s T test. Next, T24 cells were transfected with si-FGF9 and examined for (B) cell viability by MTT assay; **P<0.01, one-way ANOVA test. (C) migration capacity by wound healing assay; P<0.05, student’s T test. (D) the invasive capacity by Transwell assay, P<0.05, student’s T test.; and (E) protein levels of FGF9, ki-67, MMP-2, and MMP-9 by immunoblotting. **P<0.01, student’s T test. (F) Monocytes were isolated from peripheral blood (PBMCs) and treated with 50 ng/ml M-CSF to stimulate the monocyte differentiation into macrophages (M0). The M0 macrophages were identified as CD11b positive by flow cytometry. (G) M0 macrophages were then stimulated with 20 ng/ml IL-4 (eBioscience) for two days to induce M2 polarization and authenticated using IF staining with anti-CD11b and anti-CD206 antibodies. The inflorescence intensity is shown in the right panel. P<0.01, student’s T test. (H–K) Next, T24 cells were transfected with si-FGF9 or si-NC (negative control) and the culture medium (shown in the figures as conditioned medium, si-NC-CM and si-FGF9-CM) was collected for macrophage incubation. M0 macrophages were divided into four groups: IL-4 (M2 polarization inducing) + si-NC-CM, IL-4 (M2 polarization inducing) + si-FGF9-CM, LPS + IFNγ (M1 polarization inducing) + si-NC-CM, and LPS + IFNγ (M1 polarization inducing) + si-FGF9-CM, and examined for (H) the protein levels of CD206 and CD16 by immunoblotting. P<0.05 or P<0.01, student’s T test.; (I) the percentage of CD206 and CD16 positive cells was determined by flow cytometry; (J) the inflorescence intensity of CD206 and CD16 was measured by IF staining. The inflorescence intensity is shown in the right panel. P<0.01, student’s T test. (K) The concentrations of IL-10, Arg1, iNOS, and TNF-α in the culture medium was determined by ELISA. **P<0.01, student’s T test.