Figure 2.

CLIPR-59 contributes to TNF-α-mediated pro-apoptotic signaling. (a) HeLa cells transfected with siCLIPR-59 or siNS were stimulated with medium alone, (CHX, 10 μg/ml) or TNF-α (10 ng/ml) with CHX (10 μg/ml). At 6 h after stimulation, morphological changes were observed by light microscopy. HeLa cells transfected with siNS or siCLIPR-59 were stimulated with various amounts of TNF-α in the presence of CHX (10 μg/ml; b), or chemical cytotoxic drug, puromycin (c) or zeocin (d). At 6 h (in the case for TNF-α), 24 h (for puromycin) or 48 h (for zeocin) after stimulation, cell viability was assessed by WST assay (error bar means±S.D.; n=3). (e) HeLa cells, transfected with the plasmid for expressing GFP-tagged CLIPR-59 or GFP alone, were stimulated with TNF-α (the indicated concentration, ng/ml) with or without CHX (10 μg/ml). At 24 h after stimulation, the percentage of cell death of the transfected cells was assessed as described in Materials and Methods (error bar means±S.D.; n=3). (f) HeLa cells were transfected with the indicated expression vector plasmids. At 24 h after transfection, cell-viability, IETDase or DEVDase activity of these cells were assessed by WST assay, Caspase-8 or Caspase-3/7 Glo reagents (Promega) measuring relative fluorescent units (RFU), respectively, (error bar means±S.D.; n=3). (g) HeLa cells transfected with the indicated siRNAs directed to three different target sites on CLIPR-59 mRNA were stimulated with TNF-α (10 ng/ml) with or without CHX (10 μg/ml). At 4.5 h after stimulation, DEVDase activity was assessed as described in (f) (error bar means±S.D.; n=3). To determine the effects of siRNA treatment on the protein and mRNA expression level of CLIPR-59 were assessed by western blot using the indicated Abs and RT-PCR using primers specific for CLIPR-59 or GAPDH (lower panel), respectively