Figure 6

Transcriptional activation of Lin-28B by Stat3 contributes to transient let-7 downregulation. (a) MCF-7 cells were exposed to 10 ng/ml of OSM. The expression levels of primary let-7e (pri-let-7e) and mature let-7e (mat-let-7e) were analyzed by real-time RT–PCR and Lin-28B by western blot at the indicated time points. (b) MCF-7 cells were transfected with the Stat3C expression plasmid. The expression of Lin-28B was analyzed by western blot. (c) MCF-7/Stat3 sh cells were exposed to 10 ng/ml of OSM. The expression of Lin-28B and phophorylation of Stat3 were analyzed by western blot at the indicated time points. (d) Two Stat3 sites were identified at the upstream of the −2405 and −369 bp of the Lin-28 5′ UTR. (e) MCF-7 cells were transfected with the Flag-Stat3C expression plasmid. Binding of Stat3 to the Lin-28 5′ UTR was determined by ChIP assays using the anti-Flag antibody. (f) MCF-7 cells were exposed to 10 ng/ml of OSM and binding of endogenous Stat3 to the Lin-28 5′ UTR was determined by ChIP assays using the anti-Stat3 antibody at the indicated time points. (g) MCF-7 cells were transfected with Lin-28 siRNA. After transfection for 24 h, the cells were treated with OSM and the expression levels of E-cadherin, fibronectin, ZEB1 and Lin-28B were analyzed by western blot. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.