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. 2014 Apr 10;20(11):1693–1708. doi: 10.1089/ars.2013.5219

FIG. 1.

FIG. 1.

Nrf2 is activated in response to proangiogenic stimulation of HMEC-1. HMEC-1 were stimulated with growth factors VEGF (50 ng/ml), SDF-1α (100 ng/ml), and IL-8 (200 ng/ml) for indicated time (2, 4, 8, 12, or 24 h). (A) The level of Nrf2 protein is increased after stimulation with growth factors. (B) The effect of VEGF on Nrf2 protein tends to be dose dependent after 24 h of treatment. HMEC-1 were treated with 10 or 50 ng/ml VEGF for 4 or 24 h. (C) The level of Nrf2 protein is increased after starvation of cells in 2% FBS-containing medium for 24 h and further growth factor stimulation. (A–C) Western blot. Tubulin: control constitutive protein. SFN (2.5 μM): positive control. (D) Growth factors induce Nrf2 localization in the nucleus. Western blot. SFN: positive control. Lamin A: control constitutive protein of nuclear fraction. (E–G) The expression of Nrf2 target genes, NQO1 and HO-1, is increased in response to stimulation with growth factors. (E) 24 h, (F) different time points. RT-PCR; (G) 4 h. Western blot. Tubulin: control constitutive protein. (H) Incubation with growth factors for 2 h does not change the ROS production. DCF fluorescence measurement. (I) Akt and ERK1/2 are activated and their downstream targets S6 and p90RSK tend to be phosphorylated after 5 min of stimulation with growth factors. (J) pAkt/pS6 and pERK1/2 are decreased after incubation of VEGF-stimulated cells with wortmannin and U0126, respectively. Western blot. eIF4E: control constitutive protein; Western blot: representative experiments (nJ=experiment in duplicate); each bar represents the mean±SEM of three to four independent experiments (nH=2) performed in duplicates. *p<0.05, control versus growth factor stimulation. ERK-1/2, extracellular signal-regulated kinase-1/2; HMEC-1, human microvascular endothelial cells; HO-1, heme oxygenase-1; IL-8, interleukin-8; NQO1, NAD(P)H:quinone oxidoreductase 1; Nrf2, nuclear factor E2-related factor 2; ROS, reactive oxygen species; SDF-1, stromal cell-derived factor-1; SFN, sulforaphane; VEGF, vascular endothelial growth factor.