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. 2014 Apr 8;110(10):2569–2582. doi: 10.1038/bjc.2014.165

Figure 3.

Figure 3

Endogenous mutant p53 (R280K) supports TGF-β/SMAD-dependent Nox4 induction in MDA-MB-231 breast epithelial cells. (A) MDA-MB-231 cells were transfected with control siRNAs (50 nM) or p53-specific siRNAs (50 nM) for 72 h then simulated with TGF-β (5 ng ml−1) for 24 h. Real-time quantitative PCR analysis of Nox4 mRNA expression was determined from MDA-MB-231 cells treated as in A (n=3, in triplicate). (B) MDA-MB-231 cells were treated as in A followed by protein expression analysis by immunoblotting 40 μg of total cell lysate. The blot was sequentially probed with antibodies against Nox4, p53, phospho-SMAD3, and total SMAD3. (C) MDA-MB-231 cells were transfected with non-targeting control (50 nM) or SMARTpool Nox4-specific siRNAs (50 nM) for 72 h and either left untreated or treated with TGF-β (5 ng ml−1) for an additional 24 h. Nox4 protein expression was analysed by western blotting. Immunoblots were probed with anti-Nox4 followed by anti-GAPDH. (D) MDA-MB-231 cells were treated as described and assayed for superoxide production with superoxide-specific Diogenes reagent for 1 h (n=3, in triplicate). (E) MDA-MB-231 cells were treated with transfection reagent alone or transfected with control or p53-specific siRNAs. After 72 h, the cells were re-seeded in the upper chamber of a Matrigel transwell and incubated in the lower chamber containing RPMI-1640 medium containing TGF-β 5 ng ml−1 for 24 h. The migrating cells were counted from 10 random fields (n=3). (F) MDA-MB-231 cells were left untreated or treated with DMSO (vehicle), 616451 (10 μM), or SIS3 (10 μM) for 4 h before the addition of TGF-β (5 ng ml−1) for 24 h. Protein expression was analysed by immunoblotting 40 μg of total cell lysate and sequentially probed with the indicated antibodies. (G) Total RNA extracted from cells treated as in F was reverse transcribed for real-time quantitative PCR analysis of Nox4 mRNA expression. Results are described as relative quantification of Nox4 mRNA relative to untreated control (n=3, in triplicate). Significant values are indicated as *P-value<0.05, or **P-value<0.01.