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. 2016 Jul 6;7(47):76577–76589. doi: 10.18632/oncotarget.10428

Figure 3. Dose-depending efficacy of dasatinib on TS promoter inhibition and SRC direct involvement in TS promoter/protein modulation.

Figure 3

REN cells treatment with scalar dasatinib concentration inhibited TS expression and SRC activation already at 100 nM concentration. A similar behavior was seen for the TS-promoter (REN TS-Prom-1191 + 176 clones D7 and D9). In particular, in each clone, dasatinib reduced TS promoter mediated luciferase activity at 24 and 48 h. This effect was also confirmed at the TS protein level (A). To assess the effect of combination of dasatinib and PEM we measured the luciferase expression in five independent REN TS- Prom-1191 + 176 clones. After pretreatment for 12 hours with 0.5 μM dasatinib, all clones expressed low levels of luciferase compared to controls. Clones were subsequently incubated for 72 hours with PEM at 1 μM (PEM) either removing (rD→PEM) or maintaining (mD→PEM) dasatinib pretreatment. An incubation with 72 hours of PEM alone was in parallel carried out. In all the clones examined, mD→PEM condition was the most efficient in downregulating TS transcriptional activity (B). The expression of a dominant negative form of SRC (SRC K295M) impaired expression of luciferase under control of TS-promoter at both 24 and at 48 h post-transfection. Furthermore, the effect of SRC KD K295M was assessed also at the protein level, resulting in a reduction of the endogenous TS protein (C).