Figure 4.
shRNA-mediated silencing of STAT3 sensitizes SW480 cells to chemoradiotherapy. (a) Stable single-cell clone populations were established for each inducible shRNA vector targeting STAT3, and successful silencing was confirmed 120 hr after addition of 1 μg/ml doxycycline to induce shRNA expression and stimulation with 100 ng/ml IL-6 for 30 min using Western blotting. (b) Reduced STAT3 transcription factor activity was demonstrated using a dual luciferase reporter assay (P < 0.05). (c) Ninety-six hours after doxycycline induction, cell clones were incubated with 3 μM of 5-FU and irradiated at 1, 2, 4, 6 and 8 Gy of X-rays (without stimulation with IL-6). shRNA-mediated silencing of STAT3 led to a significantly increased treatment sensitivity (vector 1, P < 10−24; vector 2, P < 10−14; ANOVA model). All experiments were done as triplicates and showed similar results, error bars ± SEM.