Figure 2

BOT-4-one inhibits expression and phosphorylation of STAT3 in human cancer cell lines. (A) Human cancer cell lines that express constitutively-active STAT3 were incubated with either vehicle (DMSO) alone or BOT-4-one (30 µM) for 24 h. Total RNA was extracted and subjected to quantitative real-time PCR. BOT-4-one inhibited STAT3 expression in all cell lines, but inhibition of STAT1 and STAT5 expression was cell type-dependent manner. Results are shown as the mean of three independent experiments (± SD indicated by error bar). ^*P < 0.001, significant difference when the value of treatment was compared to that of the control. (B) Whole cell extracts were prepared from L540 cells after treatment for 24 h with either vehicle (DMSO) alone or BOT-4-one, and immunoblot analysis was performed with antibodies specific for the molecules indicated. BOT-4-one inhibited tyrosine phosphorylation of STATs, and its inhibitory effect on STAT3 and STAT5 was much higher than those of STAT1. GAPDH served as a loading control. (C) Cytosolic and nucleic fractions were extracted from L540 cells after treatment for 24 h with either vehicle (DMSO) alone or BOT-4-one, and immunoblot analysis was performed with phospho-STAT3 and STAT3 antibodies. BOT-4-one inhibited STAT3 activation. C, cytosolic, N, nucleic.