Figure 1

TSN selectively inhibits STAT3 activity in osteosarcoma. (a) Osteosarcoma cells were transfected with luciferase reporter gene plasmid and treated with TSN for 24 h. The results were normalized to the Renilla luciferase activity. The bars indicate the mean±s.d. Statistically significant differences (t-test), *P<0.05; **P<0.01; ***P<0.001. (b) The chemical structure of TSN. (c) After treatment with TSN for 24 h, cell extracts were prepared and applied to immunoblotting with phospho-STAT3 (Tyr-705) or phospho-STAT3 (Ser727). Actin was used as a loading control. (d) Left panel, 143B cells were cultured on gelatin-coated coverslips and treated with TSN for 24 h followed by stimulating with IL-6 (20 ng/μl) for 30 min. Anti-STAT3 antibody (green) was used to locate endogenous STAT3. Cell nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). The photographs were acquired by a confocal microscopy. Scale bar, 20 μm. Right panel, l43B cells were treated with TSN for 24 h, followed by stimulating with IL-6 (20 ng/μl) for 30 min, and the cytoplasmic and nuclear extractions were subjected to immunoblotting to detect the distribution of STAT3. (e) 143B cells were pretreated with TSN. An EMSA assay was performed to analyze STAT3 DNA-binding activity. (f) Cells were treated with TSN for 24 h. STAT3 target genes expression were analyzed by RT-PCR. (g) Cells were treated with TSN for 24 h. Cells were then lysed and applied to immunoblotting with indicated antibodies. Actin was used as a loading control. (h) 143B cells were incubated with TSN and analyzed by a quantitative ChIP assay with anti-STAT3 antibody. The bars indicate the mean±s.d. Statistically significant differences (t-test), *P<0.05; ***P<0.001.