Files in this Data Supplement:

• Supplemental Methods - (Word ) (61.5 KB)
• Figure I - (Power Point) (886 KB) Changes in the expression of t- & p-Akt, t- & p-Erk, IκBβ, HIF-1α in response to hypoxia. Serial immunoblot analysis of the downstream molecules of ILK, p-Akt, p-Erk, IκBβ, HIF-1α in time sequence (n=4).
• Figure II - (Power Point) (7.38 MB) Confirmation of kinase activity of adenoviral constructs of ILK and dead-ILK, and change of downstream target genes in immunofluorescence staining of ILK-modulated HUVECs. A, In vitro kinase assay showing the enhanced kinase activity of adv-ILK viral constructs. B, In vitro kinase assay showing the absence of kinase activity of adv-dead ILK, the kinase deficient form of ILK, viral constructs. C & D, Immunofluorescence staining of ICAM-1 and SDF-1 in ILK overexpressed normoxic HUVEC. E & F, Immunofluorescence staining of ICAM-1 and SDF-1 in ILK blocked, adv-dead ILK transfected hypoxic HUVEC.
• Figure III - (Power Point) (1.01 MB) Assay of EPC incorporation to adv-ILK transfected normoxic HUVEC or adv-dead ILK transfected hypoxic HUVEC monolayer. A, Representative figures of EPC incorporation assay after transfection of either adv-GFP or adv-ILK into normoxic HUVECs and the effect of blocking ICAM-1 and SDF-1. C : control antibody. B, Representative figures of EPC incorporation assay in adv-GFP or adv-dead ILK transfected hypoxic HUVEC monolayer. C, Manual counting of DiI-tagged EPCs in ischemic hindlimbs either injected with adv-GFP or adv-dead ILK. Ten random fields were selected in each specimen (*, p < 0.05, vs. adv-GFP injected limb; n=3).
• Figure IV - (Power Point) (3.12 MB) Recovery of perfusion in adv-GFP and adv-dead ILK injected ischemic hindlimbs. A, Representative figures of the serial LDPI showing the natural recovery of perfusion in adv-GFP injected hindlimbs, in contrast to the poor recovery of perfusion in adv-dead ILK injected ischemic hindlimb up to day 21. High perfusion is indicated by red and low perfusion by blue. B, Representative photography of the ischemic hindlimbs at 21 days after the operation. Adv-GFP transfected limbs mostly showed foot necrosis and even complete limb salvage. Adv-dead ILK transfected limbs showed absolute autoamputation in all animals.