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Blood, Vol. 112, Issue 2, 330-339, July 15, 2008
ATF4-dependent transcription is a key mechanism in VEGF up-regulation by oxidized phospholipids: critical role of oxidized sn-2 residues in activation of unfolded protein response
Blood Oskolkova et al.
112: 330
Supplemental materials for: Oskolkova et al
Files in this Data Supplement:
- Document 1. Supplemental results (PDF, 3.15 MB)
- Figure S1. The PAF-receptor does not play a role in OxPL-induced upregulation of VEGF (JPG, 22.2 KB)
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(A) HUVECs were stimulated with 130 µmol/L of OxPAPC or 0.5 µmol/L of PAF in medium 199 containing 2% FCS. (B) HUVECs were incubated with 130 µmol/L of OxPAPC with or without PAF-receptor antagonists gingkolide B (5 µmol/L) or CV-3988 (10 µmol/L). The incubation was continued for 6 hours followed by mRNA isolation and VEGF mRNA
quantification.
- Figure S2. The upregulation of VEGF by OxPAPC is not mimicked by 8-iso¬prostaglandin E2 (JPG, 16.6 KB)
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HUVECs were stimulated with 130 µmol/L of OxPAPC or 50 µmol/L of 8-iso-PGE2 in medium 199 containing 2% FCS. The incubation was performed for 6 hours.
- Figure S3. Lipopolysaccharide (LPS) does not upregulate VEGF mRNA levels in HUVECs (JPG, 15.6 KB)
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The cells were stimulated with 130 µmol/L of OxPAPC or 100 ng/mL of LPS in medium 199 containing 2% FCS for 6 hours, followed by RNA extraction and VEGF mRNA quantification.
- Figure S4. PPAR agonists do not influence VEGF mRNA expression (JPG, 20 KB)
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HUVECs were stimulated with 130 µmol/L of OxPAPC or indicated concentrations of PPAR or PPAR agonists (Wy-14643 and rosiglitazone, respectively) in medium 199 containing 2% FCS. The incubation was performed for 6 hours.
- Figure S5. Knock-down of ATF4 mRNA using RNA interference technology (JPG, 17.9 KB)
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HUVECs were transfected with siRNA against ATF4 or control siRNA. After 48 hours, the cells were stimulated with 130 µmol/L of OxPAPC in medium 199 containing 2% FCS. After 6 hours the incubation was terminated with Trizol and ATF4 mRNA quantified using real-time PCR. The data are normalized to  2-microglobulin mRNA expression levels.
- Figure S6. ATF4 binds to its predicted site in VEGF promoter (JPG, 18.3 KB)
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HeLa cells were treated with OxPAPC (130 µmol/L) for 3 hours and then processed for ChIP analysis as described in the “Materials and Methods” section. Immunoprecipitation was performed by two antibodies recognizing different epitopes within the ATF4 protein. The Figure presents results of ethidium bromide staining of products of PCR amplification of the fragments of VEGF gene. The fragment overlaps ATF4 binding site that was designated throughout the paper as “AsnSyn.” The “input” was obtained by amplification of 1% of initial unfractionated cell extract.
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