HUVECs (Thermo Fisher Scientific, Rockford, IL, USA) were used from passage 3 to passage 7 as previously described.17 (link) For hypoxia experiment, HUVECs were cultured in both normoxic and hypoxic conditions (1.5% O2 in the hypoxic chamber Xvivo System X3, Biospherix Ltd., Parish, NY, USA).
Total RNA was obtained as previously described.18 (link) Two hundred nanogram of RNA were retrotranscribed and amplified with the Express 1step SS qRT-PCR Universal (Thermo Fisher Scientific). The gene expression analysis was performed according to the “Delta delta Ct Method” normalizing to the POLR2A gene as the housekeeping gene. Taqman assays (Thermo Fisher Scientific) used were: Hs01547115_m1 (for NOVA2), Hs99999905_m1 (for GAPDH), and Hs00172187_ m1 (for POLR2A).
Western blot was performed as previously described.19 (link) Ten microgram and 35 µg of proteins were used for NOVA2 and HIF1α analysis, respectively. The following primary antibodies were used: anti-NOVA2 (Sigma-Aldrich, St Louis, MO, USA; 1:250), anti-ACTIN (AC-40, Sigma-Aldrich; 1:1,000), and anti-HIF1α (H1α 67, Novus Biologicals; 1:500).
Total RNA was obtained as previously described.18 (link) Two hundred nanogram of RNA were retrotranscribed and amplified with the Express 1step SS qRT-PCR Universal (Thermo Fisher Scientific). The gene expression analysis was performed according to the “Delta delta Ct Method” normalizing to the POLR2A gene as the housekeeping gene. Taqman assays (Thermo Fisher Scientific) used were: Hs01547115_m1 (for NOVA2), Hs99999905_m1 (for GAPDH), and Hs00172187_ m1 (for POLR2A).
Western blot was performed as previously described.19 (link) Ten microgram and 35 µg of proteins were used for NOVA2 and HIF1α analysis, respectively. The following primary antibodies were used: anti-NOVA2 (Sigma-Aldrich, St Louis, MO, USA; 1:250), anti-ACTIN (AC-40, Sigma-Aldrich; 1:1,000), and anti-HIF1α (H1α 67, Novus Biologicals; 1:500).
