Figure 6
Accumulation of p65 facilitates EMT program. (a) MCF-10A-NC and MCF-10A-6SA cells were treated with TNFα (10 ng/ml) for 45 min followed by nuclear fractionation analysis with immunoblots. Lamin B and β-tubulin served as internal control for nucleus and cytoplasm, respectively (left). Right: quantification analysis of band density of nuclear p65 by ImageJ and normalized to that of lamin B. ***P<0.001 as compared with MCF-10A-NC cells in each group. (b) MCF-10A-NC and MCF-10A-6SA cells were transfected with luciferase reporter vector driven by six repeated κB reaction elements followed by TNFα stimulation and the transcriptional activity was determined by measuring the luciferase activity. **P<0.01 and ***P<0.001 as compared with MCF-10A-NC cells in each group. (c-e) MCF-10A cells were stably infected with Siren empty vector or two shRNAs targeting p65 followed by EMT induction with Snail overexpression. (c) Immunoblots for indicated proteins. (d) Cell migration ability were analyzed with Transwell assay. Representative images were shown in d, Scale bar represents 20 μm. (e) Quantitative analysis of the migrated cell numbers in five different fields. ***P<0.001. (f) MCF-10A cells were stably infected with pMSCV empty vector or Flag-p65 overexpression vector. Cell lysates were analyzed with indicated proteins by immunoblots. (g–i) 10A-6SA cells were transfected with siNC or siRNAs targeting p65. (g) Immunoblots analysis of indicated proteins. (h) Migration ability of siNC and sip65 cells were analyzed by Transwell assay. Representative images of migrated cells were shown in h, Scale bar represents 20 μm. (i) Quantitative analysis of the migrated cell numbers in five different fields. ***P<0.001 as compared with siNC cells.
