Bystander effects generated in MDA-MB-231 cells associated with TNFα production and TNF-receptor I expression. Bystander effects generated by adenine nucleotide translocator (ANT) 2 shRNA in MDA-MB-231 cells were associated with TNFα production and TNF-receptor I (TNFRI) expression. (a) Intracellular staining of TNFα, IFNγ and IL-12 p40. Cells were transfected with ANT2 shRNA-1. After 48 hours of incubation, the transfected cells were treated with brefeldin A for the next 6 hours. The cells were harvested, fixed in paraformaldehyde and then stained with phenylethylene-conjugated anti-TNFα, IFNγ or IL-12 p40 as well as anti-mouse IgG antibodies (negative control). Intracellular levels of TNFα, IFNγ and IL-12 p40 were analyzed by flow cytometry. (b) RT-PCR and fluorescent-activated cell sorter analysis for detecting the expression of TNFRI at transcriptional and translational levels. Cells were transfected with ANT2 shRNA-1. After 24 hours of incubation, total RNA was extracted and subjected to RT-PCR using specific primers for human TNFRI and β-actin. The RT-PCR products were analyzed by 1% agarose gel electrophoresis. In addition, the surface expression of TNFRI was measured by flow cytometry after staining cells with phenylethylene-conjugated anti-TNFRI antibody. (c) Partial neutralization of bystander effect mediated by anti-TNFα antibody. Cells were transfected with ANT2 shRNA-1. After 48 hours of incubation, the supernatants were collected and then mixed with or without TNFα antibody before transferring into nontransfected cells. These nontransfected cells were cultured for the next 24 hours and stained with annexin V–fluorescence isothiocyanate (FITC) and propidium iodide (PI) for flow cytometric analysis.