FIGURE 6.

The STAT3 small molecule inhibitor LLL12 blocks STAT3 phosphorylation in a dose- and time-dependent manner. A, Hep3B and SNU-398 cells were cultured in serum-free medium for 24 h and then were pretreated with various concentrations of LLL12 (0.5–5 μm) for 2 h followed by IL-6 for 30 min. Phosphorylated STAT3 and total STAT3 were detected by Western blot. B, Hep3B and SNU-398 cells were cultured under the same conditions and then were treated with 5 μm LLL12 for different time points (0–2 h). After pretreatment, IL-6 was added to the cultured cells. Phosphorylated STAT3 and total STAT3 were detected by Western blot. C, Hep3B cells were cultured in serum-free medium for 24 h and then were pretreated with 5 μm LLL12 for 2 h. After the 2 h pretreatment, medium was discarded, and fresh medium without LLL12 was added. After the indicated incubation (0–24 h), the cells were treated with IL-6 for 30 min. Phosphorylated STAT3 and total STAT3 were detected by Western blot. D, Hep3B cells were cultured in serum-free medium for 24 h and then were pretreated with 5 μm LLL12 for 2 h. After 2 h of pretreatment, the medium was discarded, and fresh medium without LLL12 was added. Cycloheximide was also added into the medium to block protein synthesis. After 12 h, the cells were treated with IL-6 for 30 min. Phosphorylated STAT3 and total STAT3 were detected by Western blot.