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. 2011 Dec 27;6(12):e29680. doi: 10.1371/journal.pone.0029680

Figure 4. L-NAME treatment induces HIF-1α nuclear accumulation.

Figure 4

(A) HIF-1α protein levels were detected by western blotting of nuclear extracts from control HUVECs (lane 1) or from HUVECs treated with L-NAME for 48 h (lane 2). Shown is a representative blot of 4 comparable experiments. HIF-1α migrates as a doublet with apparent molecular weight of 118 and 120 kDa. (B) Densitometric analysis of nuclear HIF-1α protein levels. *p<0.05; t test; n = 4. (C) HIF-1α RNA levels were measured by RT-qPCR and normalized to the level of the housekeeping gene 18S. No significant differences between control and L-NAME treated cells (t test; n = 3). (D) VEGF protein levels were detected by ELISA measurement in conditioned media collected from HUVECs transfected with the empty vector (pcDNA3) or with the expression vector ΔARNT, and treated with L-NAME for the 48 h following transfection. Results are expressed as pg of VEGF normalized to the cell protein content (pg/mg protein). **p<0.01 vs untreated cells transfected with pcDNA3; ***p<0.001 vs L-NAME treated cells transfected with pcDNA3 (One-way ANOVA with Bonferroni's test; n = 3). (E) HUVECs were transfected with pcDNA3 or ΔARNT, and treated with L-NAME for the 48 h following transfection when indicated. Chemotaxis experiments were then performed using 25 ng/ml VEGF as attractant. Results are expressed as the number of migrating cells. #p<0.001 vs basal migration in untreated pcDNA3 cells; §p<0.001 vs VEGF-induced migration in untreated pcDNA3 cells; ***p<0.001 vs basal migration in pcDNA3 cells treated with L-NAME; °°°p<0.001 vs VEGF-induced migration in pcDNA3 cells treated with L-NAME; no significant differences between untreated pcDNA3 and ΔARNT transfected cells and between untreated and L-NAME treated ΔARNT tranfected cells (One-way ANOVA with Bonferroni's test, n = 10).