Figure 4

Stat3 activation mediates OSM-induced repression of let-7 and miR-200. (a) MCF-7 cells were treated with 10 ng/ml of OSM and then p-Stat3, p-c-Jun and c-Fos were analyzed by western blot at the indicated time points. (b) MCF-7 cells were stably transfected with the plasmid expressing Stat3-specific shRNA (MCF-7/Stat3 sh). The expression of Stat3 was determined by western blot. (c) The transfected cells were treated with 10 ng/ml of OSM for 4 days. The expression levels of E-cadherin, fibronectin, ZEB1 and p-Stat3 were analyzed by western blot. (d) MDA-231 cells were stably transfected with the plasmid expressing Stat3 shRNA (MDA-231/Stat3 sh). The expression levels of E-cadherin, fibronectin, ZEB1 and Stat3 were analyzed by western blot. (e, f) MCF-7/Stat3 sh cells were treated with 10 ng/ml of OSM for 5 days. The invasive activities of the cells were determined by Matrigel invasion assays after 10 h (MDA-231/Stat3 sh) or 48 h (MCF-7/Stat3 sh) of culture. The invading cells were stained (e) and quantified (f). (g) MCF-7 cells were transfected with the plasmid expressing Stat3C and the expression of let-7b/d/e/g and miR-200b/c was analyzed by real-time RT-PCR after transfection for 48 h. (h, i) The expression levels of let-7e and miR-200c in MCF-7/Stat3 sh cells were analyzed by real-time RT-PCR at the indicated time points after OSM induction. (j) MCF-7 cells were cotransfected with the plasmids containing HMGA2 5′ and 3′ UTRs, ZEB1 3′ UTR and Stat3C. Luciferase activities were measured after transfection for 36 h. Error bars indicate s.e. (N⩾3). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.