Overexpression of nucleolin protects mice from a lethal Fas activation through nucleolin-Fas complex. (A) Mice were hydrodynamically transfected with a vector control or a plasmid expressing DDK-tagged full-length nucleolin. The mice were challenged 24 hours later with a lethal dose of Jo2 agonistic anti-Fas antibody (2 µg/g weight) and monitored for survival for up to 8 hours post-challenge. The survival rate of nucleolin-transfected mice was significantly higher than mice transfected with vector alone (P < .006; log rank Mantel-Cox test). Representative data from 3 different experiments are shown. (B) Gross examination of vector and nontransfected livers challenged with Jo2 revealed massive hemorrhaging as indicated by darkening and swelling. Nucleolin-expressing livers challenged with Jo2 showed decreased hemorrhaging. (C) Livers were harvested, resected, and stained with hematoxylin and eosin (upper panel), or were analyzed using cleaved caspase-3 antibody, cleaved PARP antibody, or TUNEL assay to evaluate apoptosis. The images were captured by the Olympus BX41 (Olympus) UPlan FL N 40×/0.75 objective. Images were acquired with DP Controller (Olympus) with a −2 exposure adjustment for TUNEL staining with a fluorescein isothiocyanate filter (Olympus). Adobe Photoshop PS2 was used for further image enhancement of green fluorescent protein with a +30 brightness for all 4 panels equally. (D) Homogenized vector-tramsfected, nucleolin-transfected, a nontransfected Jo2-challenged and Jo2-unchallenged liver samples were subjected to lysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and IB analysis of nucleolin, caspase-8, caspase-3, PARP, Bcl-2, and β-actin. (E) Mice were transfected with vector control, DDK-tagged full-length nucleolin, or DDK-tagged mutant lacking the Fas-nucleolin binding domain NR123 plasmids. Mice were challenged with a lethal dose of Jo2-agonistic anti-Fas antibody (.5 µg/g weight) and monitored for survival for up to 8 hours post-challenge. On gross examination, nontransfected, vector-transfected, and NR123-transfected livers challenged with Jo2 exhibited massive hemorrhaging as shown by darkening and swelling. Nucleolin livers challenged with Jo2 showed decreased hemorrhaging as shown by light spotting. We confirmed expression of nucleolin and NR123 by IB (data not shown). The survival rate was significantly higher for nucleolin-transfected mice than for mice transfected with vector alone or nonbinding mutant NR123 (P < .003; log rank Mantel-Cox test). Combined data from 2 independent experiments are shown.