TLR4 Ligand/H2O2 Enhances TGF-β1 Signaling to Induce Metastatic Potential of Non-Invasive Breast Cancer Cells by Activating Non-Smad Pathways
Figure 3
H2O2 and LPS enhance the sustained activation of Smad pathway.
(A) MCF-7 cells were stimulated with TGF-β1 (5 ng/ml). Phospho-Smad2, Smad2, phospho-Smad3 and Smad3 were detected by Western blot at the indicated time points. (B) MCF-7 cells were cultured in absence or presence of TGF-β1, H2O2 (50 µM), and LPS (100 ng/ml) for 7 days. Phospho-Smad2, Smad2, phospho-Smad3 and Smad3 were detected by Western blot. (C) MCF-7 cells were cultured in presence of TGF-β1/H2O2/LPS. Phospho-Smad2, Smad2, phospho-Smad3, Smad3, Smad7, and β-actin were detected by Western blot at the indicated time points. Smad4 in nuclear extract was detected by Western blot. Lamin B1, a nuclear protein, in nuclear extract was used as control. (D) MCF-7 cells were cultured in absence or presence of TGF-β1/H2O2/LPS (left) for the indicated time. Or the cells were cultured for 6 days in absence or presence of TGF-β1, H2O2, and LPS (right). The expression of SMAD7 (D) and SNAI2 (E) were detected by real-time RT-PCR. P values, *P<0.05, **P<0.01.