Technical Reference #3259

3259.

The Interferon Stimulated Gene 54 Promotes Apoptosis Marcin Stawowczyk; Sarah Van Scoy; K. Prasanna Kumar; and Nancy C. Reich, Stony Brook University, Journal of Biological Chemistry, 286(3259), (2011)
Link To Paper

Abstract:
The ability of interferons (IFNs) to inhibit viral replication and cellular proliferation is well established; but the specific contribution of each IFN stimulated gene (ISG) to these biological responses remains to be completely understood. In this report we demonstrate that ISG54; also known as IFN-induced protein with tetratricopeptide repeats 2 (IFIT2); is a mediator of apoptosis. Expression of ISG54; independent of IFN stimulation; elicits apoptotic cell death. Cell death and apoptosis were quantified by propidium iodide uptake and annexin-V staining; respectively. The activation of caspase-3; a key mediator of the execution phase of apoptosis; was clearly apparent in cells expressing ISG54. The anti-apoptotic B cell lymphoma-xl (Bcl-xl) protein inhibited the apoptotic effects of ISG54; as did the anti-apoptotic adenoviral E1B 19K protein. In addition; ISG54 was not able to promote cell death in the absence of pro-apoptotic Bcl family members; Bax and Bak. Analyses of binding partners of ISG54 revealed association with two homologous proteins; ISG56/IFIT1 and ISG60/IFIT3. In addition; ISG60 binding negatively regulates the apoptotic effects of ISG54. The results reveal a previously unidentified role of ISG54 in the induction of apoptosis via a mitochondrial pathway; and shed new light on the mechanism by which IFN elicits anti-viral and anti-cancer effects.

Materials & Methods:
Microscopy—Immunofluorescence was performed after cell fixation with 4% paraformaldehyde and permeabilization in 0.2% Triton X-100. Cells were incubated with primary antibody followed by secondary antibody conjugated to TRITC. Cells were visualized with Zeiss Axiovert 200M and Axiovision Version 4.5. Live cell imaging was performed with cells seeded on glass-bottom plates (Mattek Corp.). Mitochondria were visualized by incubation with 500 nM MitoTracker Orange CMTMRos before imaging (Invitrogen). The Zeiss Tempcontrol 37–2 Digital and CTI Controller 3700 was used with the Zeiss LSM 510 laser scanning microscope system (Zeiss) and an alpha Plan-FLUAR 100 /1.45 objective. Live cell images were captured using Zeiss LSM 5 Pascal imaging software.

Microscopic Technique
Immunofluorescence, laser scanning microscope system

Cell Type(s)
kidney cells