Figure 3. Inhibition of miR1-38 restores normal function to cytokine-treated ECs.

A) EA.hy926 ECs were transfected with the S100A1–3’UTR luciferase reporter construct and incubated with either antagomir-138 or control and stimulated for 24h with cytokines or CoCl₂ as indicted prior to luciferase assay. *, P<0.02 vs untreated. B) Primary HMVECs were treated with Ang II ± antagomir-138 for times indicated and S100A1 and phospho-T495 eNOS levels were assessed by Immunoblot analysis. β-actin was assessed as loading control. Representative images are shown. Experiment was repeated twice more, for quantification see Figure S9. C) HMVECs were incubated with cytokines for 24h and co-incubated with either antagomir-138 or control, then treated ± VEGF (50 ng/ml) as indicated for a further 24h. eNOS produced nitric oxide levels in the supernatant were measured using a nitrate/nitrite assay. *, P<0.05 vs untreated. # P<0.05 vs untreated, no VEGF; ✠, P<0.05 vs untreated + VEGF. Experiment was performed 3 times, each at least in duplicate. D) Primary HMVEC were incubated with antagomir-138 or control and treated with Ang II for 24h. Next day later cells were infected (MOI=17) with either control Adenovirus or Adenovirus expressing S100A1. 24h later cells were seeded onto Matrigel matrix. Images of EC tube formation were taken 24h later and digitized using Image J (Original pictures of EC tube formation are included as supplemental Figure S11. Representative images are shown. The experiment was done 3 times, each in duplicate.