Pper chambers and treated with the indicatives for 24 hours. Invading cells were stained with crystal violet and then cell numbers were measured. 0.05. (c) MDA-MB-231 cells were cultured in soft agars and treated with the indicatives for 15 days. Colonies were then stained with crystal violet. 0.05.effects of SH003 on MDA-MB-231 cells, we next examined intracellular signaling pathway. Cells were treated with each extract at 50 g/mL (Figure 5(a)) or 500 g/mL (Figure 5(b)) for 15 minutes and subjected to the western blots. While phosphorylation of EGFR and SRC was partly reduced by 50 g/mL of SH003 or each component (Am, Ag, and Tk), STAT3 phosphorylation was strongly and selectively inhibited by SH003. Furthermore, STAT3 phosphorylation was also selectively inhibited by SH003 at 500 g/mL, while each component at 500 g/mL did not repress it. Therefore, we assumed that SH003 selectively blocked STAT3 phosphorylation.Next, we examined whether SH003 affects transcriptional activities of STAT3. When STAT3 nuclear translocation was examined, SH003 at 500 g/mL blocked nuclear translocation of phosphorylated STAT3 (Figure 5(c)). In the luciferase assays, SH003 at 500 g/mL also inhibited transcriptional activities of STAT3 in constitutively active STAT3- (CASTAT3-) overexpressed 293T cells, while STAT3 silencing (STAT3i) in 293T cells reduced STAT3-dependent transcriptional activities (Figure 5(d), left). Likewise, SH003 reduced STAT3 transcriptional activities in MDA-MB-231 cells where STAT3 is constitutively activated, which was similar to the effect of STAT3 silencing on STAT3 transcriptional activityMediators of Inflammation50 g/mL Control Control SH003 Am Ag Tk 500 g/mL SH003 Am Ag Tkp-EGFR EGFR p-JAK1 p-JAK2 p-SRC SRC p-AKT AKT p-ERK ERK p-STAT3 STAT3 Tubulinp-EGFR EGFR p-JAK1 p-JAK2 p-SRC p-STAT3 SRC p-AKT AKT p-ERK ERK p-STAT3 STAT3 TubulinControlSH(a)(b)MergeTOPRO-(c)8 Rel. luc. activity Rel. luc. activity1.0.0 STAT3i– — SH– SHCA-STAT3 p-STAT-lucp-STAT-luc(d)Figure 5: SH003 selectively inhibits STAT3 phosphorylation and transcriptional activity. ((a) and (b)) MDA-MB-231 cells were treated with the indicatives at 50 or 500 g/mL for 15 minutes and then subjected to western blots with the antibodies indicated. Tubulin was used for the internal control. (c) Cells were treated with the indicatives for 6 hours and then stained with anti-p-STAT3 antibody (green) and TOPRO-3 (blue). 20x objectives. A scale bar indicates 10 m. (d) Representative data for the luciferase assays. 293T (left) and MDA-MB-231 (right) cells were transfected with the indicatives and then treated with each extract for 24 hours. Experiments were performed in triplicate.Bethanechol chloride Bars indicate means and standard deviations.Encorafenib 0.PMID:23398362 05.(Figure 5(d), right). Therefore, our data indicate that SH003 selectively inhibits STAT3 activity. 3.6. SH003 Inhibits Expression of STAT3 Target Genes and IL-6 Production. As SH003 suppressed STAT3 activation, wenext examined whether SH003 affects expression patterns of STAT3-dependent genes. SH003 at 500 g/mL inhibited protein expression levels of STAT3-dependent genes such as Cyclin D, MMP-9, VEGF, and Survivin, while 50 g/mL of SH003 only decreased levels of Cyclin D1 and MMP-STAT3i50 g/mL 500 g/mLMediators of InflammationControl Am AgControl Am AgTk SHTk SH1.IL-6 relative expression (mRNA)Cyclin DCyclin DMMP-9 VEGF Survivin Tubulin(a) (b)MMP-9 VEGF Survivin Tubulin0.0 Control(c)SH100 1.5 IL-6 concentration (fold change)STAT3 on IL-6 promote.