Smart pools of mouse Bim small interfering

RNA (siRNA) duplexes and nontargeting control duplexes were purchased from Dharmacon (ON-TARGETplus SMARTpool); the Lipofectamine RNAiMAX transfection reagent was obtained from Invitrogen. For siRNA transfection, cells were reverse-transfected with 10 nM siRNA with Lipofectamine in the Opti-MEM medium according to the manufacturer’s instructions. Effective knockdown was verified by quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblotting after different times selleck chemicals (Supporting Fig. 1). Other experimental procedures are described in detail in the supporting information. These include the mice, preparation of total, cytosolic, und mitochondrial lysates, western blotting, quantification of neuroblastoma 2A

(N2A) FasL, quantification of V1q TNFα-neutralizing antibody, DEVDase assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay, Cell Death Detection enzyme-linked immunosorbent assay (ELISA), RNA isolation, complementary DNA synthesis and qRT-PCR, and cytochrome c ELISA. We previously reported that FasL induces the apoptosis of collagen-cultured primary murine hepatocytes via the type I signaling pathway, but only to a moderate extent.12 In this study, we focused on the crosstalk of FasL with the proinflammatory cytokine TNFα. We preincubated collagen-cultured primary murine hepatocytes with 25 ng/mL TNFα for selleck kinase inhibitor 12 hours, and this was followed by a treatment with 50 ng/mL FasL for 6 hours. As expected, untreated and TNFα-treated hepatocytes showed a typical binuclear morphology and no signs of cell death over an incubation period of 18 hours (Fig. 1A). In contrast, as previously Dimethyl sulfoxide reported, cells treated with FasL for 6 hours showed hallmarks of apoptosis such as cell shrinkage

and plasma membrane blebbing.12 When the cells were preincubated with TNFα for 12 hours before the FasL treatment, they underwent a significantly higher degree of apoptosis (Fig. 1A). These findings could be confirmed by the measurement of the effector caspase-3/caspase-7 activity in response to the different treatments. As shown in Fig. 1B, the longer the hepatocytes were cultured (12, 24, or 48 hours), the more caspase-3/caspase-7 activity they displayed with a 6-hour FasL treatment. If during this culturing the cells were exposed to TNFα, the caspase-3/caspase-7 activities further increased and were consistently higher than those with FasL alone. Importantly, a minimum preincubation time of approximately 2.5 to 3 hours was needed for TNFα to exert its sensitization on FasL-induced caspase-3/caspase-7 activation, and this indicated that the TNFα effect was not immediate (Fig. 1C). We also tested the dose dependence of the sensitization and found that varying the TNFα concentrations from 10 to 50 ng/mL did not modulate the preincubation time required for sensitization (Supporting Fig. 2).