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. Author manuscript; available in PMC: 2015 Mar 18.
Published in final edited form as: FEBS Lett. 2014 Jan 31;588(6):906–914. doi: 10.1016/j.febslet.2014.01.033

Figure 2. Pro-inflammatory cytokines decrease S100A1 via increased HIF1-α and miR-138 levels in ECs.

Figure 2

A) Primary HMVECs were incubated with Ang II, Et-1, TNFα or CoCl2 (250 μmol/L) for 24h before extract preparation and SDS-PAGE analysis for HIF1-α. Representative immunoblot is shown. Experiment was repeated twice more, for quantification see Figure S2. B) Expression levels of miR-138 were assessed by qPCR in EC extracts subjected to 24h treatment with Angiotensin II (Ang II, 10 nmol/L), Endothelin-1 (Et-1, 10 nmol/L) or human TNFα (TNF, 10ng/ml). Expression levels of the small nuclear RNA U6 were assessed in parallel. U6 levels were not changed by cytokine treatment. *, P<0.05 vs untreated. The experiment was performed 4 times, each at least in duplicate. C) EA.hy926 ECs were transfected with the WT S100A1 luciferase reporter construct or the miR-138 binding site deleted reporter (Δ-miR-138) and treated for 24h with Ang II, Et-1 or TNFα, prior to luciferase assay. *, P<0.05 vs untreated. Experiment was done 3 times, each in triplicate. D) EA.hy926 ECs were transfected with the S100A1–3’UTR reporter gene construct and co-transfected with either siRNA against HIF1-α (Dharmacon) or scramble control and subsequently treated with cytokines as indicated. Treatment with CoCl2, a chemical inducer of the hypoxia response was included as a control. *, P<0.02 vs untreated. Experiment was done 3 times, each at least in duplicate.