Fig. 4. PBPs enhance autocrine production of TNF-α, which is required for the necroptosis of breast cancer cells. (a) Viability and (b) ROS generation in MCF-7 cells pretreated (1 h) with neutralizing antibody (5 μg/ml) against TNF-α prior to treatment with PBPs (100 μg/ml or 200 μg/ml). (c) mRNA expression levels of TNF-α and TNFR1 in MCF-7 cells treated with PBPs for 24 h, as determined by quantitative real-time PCR. (d) Detection of TNFR1 expression (green) using immunohistochemical assays of MCF-7 cells incubated with PBS (control) or stimulated with PBPs (200 μg/ml) for 48 h. Cell nuclei were counterstained with PI (red).