FJX1 stabilized HIF1-α contributes to increased endothelial tube formation <i>in vitro</i>.

<p>(<b>A</b>) Representative western blot of HIF1-α protein in SW480, HEK293T, and KM12C cells expressing vector (V) or FJX1 (FJ). β-actin served as the loading control. (<b>B</b>) Relative fold change in HIF1-α mRNA expression in SW480^FJX1MYC (FJX1) as compared to SW480^VEC (VEC). (<b>C</b>) Relative number of HMEC-1 tube structures as compared with SW480^VEC (VEC) UT after treatment with conditioned media from SW480 cell derivatives as noted. UT = untreated. siSCR = treated with scrambled siRNA. siHIF1 = treated with HIF1-α siRNA. (<b>D</b>) Representative western blot of HIF1-α and FJX1 protein in HEK293T cells transiently transfected with VEC, FJX1, or HA-HIF1-α (<b>E</b>) Relative number of HMEC-1 tube structures after treatment with conditioned media from vector, FJX1 or HIF1-α transduced HEK293T cells including fractionation of the media. RET = retained on column; FT = flow through. (<b>F</b>) Representative western blot of HEK293T cells transfected with HA-HIF1-α and VEC or FJX1 cultured in normoxia or hypoxia (4 hours) with or without treatment of MG132 (50 uM 2 hrs). (<b>G</b>) Representative western blot of HEK293T cells transfected with HA-HIF1-α and VEC or FJX1 pre-cultured in hypoxia (2 hrs) and treated with cycloheximide (100 uM) for the indicated time in minutes. Quantification is graphed on the right. (<b>B/C/E</b>) Significance was determined by ANOVA, each data point represents a biological replicate, and bars and whiskers represent mean and standard error of replicate means, respectively **P<0.005, ***P<0.0005.