Figure 2
Figure 2. RhoJ is a novel target of ERG that is implicated in EC lumen formation. HUVECs were transfected with indicated siRNA (40 nmol/L). After 48 hours of incubation, RNA was extracted for QPCR. (A) Evaluation of RhoJ expression at the RNA level in ERG siRNA–treated HUVECs. HUVECs were transfected with ERG siRNA 1-4 or nontargeting control siRNA (40 nmol/L). The mRNA levels of RhoJ within the same samples were assessed by QPCR using specific primers for RhoJ. TATA-box binding protein was used as internal control for normalization (n = 3). *P < .05. (B) Cell extracts were prepared from HUVECs transfected with either nontargeting control siRNA or ERG siRNA 4 (40 nmol/L). Equal amounts of total proteins were separated on PAGE gel. Protein levels of RhoJ and the loading control tubulin were determined by Western blot analysis. Representative Western blot analysis shows protein bands of RhoJ and tubulin. Quantitation was done by densitometric analysis (n = 3). *P < .05. (C) Schematic diagram of ERG-binding sites within the proximal promoter of the RhoJ gene. The 1.3-kb upstream promoter region of RhoJ was analyzed for putative ERG-binding site based on the known ERG consensus-binding site derived from previously characterized ERG target genes. Blue boxes indicate the putative ERG-binding sites. The bidirectional arrows marked the target regions for ChIP assays. (D-E) HUVECs were cotransfected with RhoJ promoter luciferase reporter and pCI expressing vector encoding indicated cDNA for selected ETS factors or the empty vector. After 24 hours of incubation, cells were lysed and cell lysates were analyzed for luciferase activity. The data are shown as relative luciferase activity compared with cotransfection with empty expression plasmid. (F) ChIP assay of the proximal RhoJ promoter using HUVECs. An ERG polyclonal Ab was used to precipitate the cross-linked DNA. QPCR analysis of the input in the absence of any IgG (no-Ab) and in the presence of nonspecific normal IgG (control IgG) or ERG antibody (ERG) after immunoprecipitation (IP) was performed using PCR primers corresponding to the region encoding 3 ERG putative binding sites within the proximal RhoJ promoter. The data are presented relative to control IgG. (G) Promoter transactivation assays were repeated by cotransfecting pCI-ERG with either RhoJ mutants or the wild-type promoter. The results are shown relative to control pCI-treated samples (n = 3). *P < .05.

RhoJ is a novel target of ERG that is implicated in EC lumen formation. HUVECs were transfected with indicated siRNA (40 nmol/L). After 48 hours of incubation, RNA was extracted for QPCR. (A) Evaluation of RhoJ expression at the RNA level in ERG siRNA–treated HUVECs. HUVECs were transfected with ERG siRNA 1-4 or nontargeting control siRNA (40 nmol/L). The mRNA levels of RhoJ within the same samples were assessed by QPCR using specific primers for RhoJ. TATA-box binding protein was used as internal control for normalization (n = 3). *P < .05. (B) Cell extracts were prepared from HUVECs transfected with either nontargeting control siRNA or ERG siRNA 4 (40 nmol/L). Equal amounts of total proteins were separated on PAGE gel. Protein levels of RhoJ and the loading control tubulin were determined by Western blot analysis. Representative Western blot analysis shows protein bands of RhoJ and tubulin. Quantitation was done by densitometric analysis (n = 3). *P < .05. (C) Schematic diagram of ERG-binding sites within the proximal promoter of the RhoJ gene. The 1.3-kb upstream promoter region of RhoJ was analyzed for putative ERG-binding site based on the known ERG consensus-binding site derived from previously characterized ERG target genes. Blue boxes indicate the putative ERG-binding sites. The bidirectional arrows marked the target regions for ChIP assays. (D-E) HUVECs were cotransfected with RhoJ promoter luciferase reporter and pCI expressing vector encoding indicated cDNA for selected ETS factors or the empty vector. After 24 hours of incubation, cells were lysed and cell lysates were analyzed for luciferase activity. The data are shown as relative luciferase activity compared with cotransfection with empty expression plasmid. (F) ChIP assay of the proximal RhoJ promoter using HUVECs. An ERG polyclonal Ab was used to precipitate the cross-linked DNA. QPCR analysis of the input in the absence of any IgG (no-Ab) and in the presence of nonspecific normal IgG (control IgG) or ERG antibody (ERG) after immunoprecipitation (IP) was performed using PCR primers corresponding to the region encoding 3 ERG putative binding sites within the proximal RhoJ promoter. The data are presented relative to control IgG. (G) Promoter transactivation assays were repeated by cotransfecting pCI-ERG with either RhoJ mutants or the wild-type promoter. The results are shown relative to control pCI-treated samples (n = 3). *P < .05.

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