Induction of VEGF by OxPLs is mediated by the ATF4 branch of UPR. (A,B) Correlation between the expression levels of VEGF and ATF3 (A) or TRB3 (B) in cells treated with various oxidized and unoxidized phospholipids. The data are collected from multiple experiments using different concentrations and molecular species of phospholipids. In all cases, the incubation was performed for 6 hours. Experiments using inhibitors or siRNA treatment were excluded. (C) HUVECs were transfected with siRNA against ATF4. Forty-eight hours later, the cells were stimulated for 6 hours with 130 μM OxPAPC in medium 199 containing 2% FCS. The incubations were terminated by Trizol, followed by quantification of VEGF mRNA. The data are normalized to the levels of β₂-microglobulin mRNA. (D) The experiment was performed as in panel C, but using HAECs. Incubation was performed for 4 hours. (E) HUVECs were pretreated for 30 minutes with 75 μM salubrinal followed by incubation with 130 μM OxPAPC in the presence of salubrinal in medium 199 containing 2% FCS. After 6 hours, the incubation was terminated by Trizol, and the levels of VEGF mRNA were quantified by RT-qPCR. Combined data from 2 independent experiments are shown. (F) HUVECs were incubated with 130 μM OxPAPC in medium 199 containing 2% FCS. After indicated time intervals, the cells were scraped into Laemmli buffer and analyzed by Western blotting for phosphorylated eIF2α. Afterward, the same blots were stained for total eIF2α. Intensity of immunostaining was quantified using chemiluminescent scanner. The normalized signals do not reflect real ratio of phosphoprotein and total protein due to different sensitivities of antibodies and various exposure times. (G) HUVECs were stimulated with OxPAPC (130 μM) resuspended in medium 199 containing 2% FCS. After indicated time intervals, the cells were scraped into Laemmli buffer and analyzed by Western blotting using antibodies against ATF4. (H) HUVECs were treated with OxPAPC (130 μM) or tunicamycin (3 μg/mL) for 3 hours and then processed for ChIP analysis as described in “Chromatin immunoprecipitation.” The figure presents results of ethidium bromide staining of products of PCR amplification of the fragments of VEGF and asparagine synthetase genes. The fragments overlap ATF4-binding sites within the VEGF (“AsnSyn” and “CHOP-1”)²⁶  and asparagin synthetase (NSRE-1)²⁷  genes. The antibodies used for ChIP are indicated on the top. The “input” was obtained by amplification of 1% of initial unfractionated cell extract.