Cc 2159

HUVECs were purchased from Lonza (cc-2159) and cultured in EC growth medium EGM-2 (cc-3162, Lonza). HUVECs were plated on 12-well plates at 60, 000/well or 150, 000/well and allowed to grow to 80%−90% confluence under growing conditions or 70%−80% for transfection. Cells were stimulated with or without H2O2 (Sigma) at indicated dosage or camptothecin (CPT, 10 μM, C9911, Sigma) for various times, according to the experiment: Western blot, 2, 4, 8 or 16 hours; Real-time PCR, 1, 4 or 12 hours; Tunnel staining, 4 hours; senescence β-Galactosidase staining; 1 hour. Lipofectamine 2000 transfection reagent (11668019, Invitrogen) was used for transfection, following the manufacturer’s instructions. MiRNA negative control (NS-m) (AM17110, Ambion) or pre-miR-181b (181b-m) (PM12442, Ambion) were transfected at 10 nM, and miRNA inhibitor negative control (NS-i) (AM17010, Ambion) or miR-181b inhibitor (181b-i) (AM12442, Ambion) were transfected at 100 nM. SiRNA control (Ctrl si) (AM4636) and validated MEKK3 siRNA (MEKK3 si) (AM51331) from Invitrogen were transfected at 20 nM. For co-transfection studies, NS-i (100 nM), 181b-i (100 nM), Ctrl si (20 nM), or MEKK3 si (20 nM) were transfected as indicated in respective experiments.

HUVECs were purchased from Lonza (cc-2159) and cultured in EC growth medium EGM-2 (cc-3162). Cells were plated on 12-well plates at 60,000/well and allowed to grow to 80%–90% confluency. In some experiments, HUVECs were stimulated with 10 ng/ml of recombinant human TNF-α (210-TA/CF, R&D Systems) for 4 h. After stimulation, HUVECs were harvested for RNA isolation using TRIzol reagent (15596018, Invitrogen). Cells were passaged less than five times for all experiments. Primary lung and liver ECs from miR-181a2b2^flox/flox, miR-181a2b2 knock out (miR-181a2b2^−/−), or ECs-specific miR-181a2b2 deficient mice (miR-181a2b2^iECKO) mice were isolated and cultured as described in our previous works [10 (link),12 ]. Lung or liver ECs were stimulated with or without 20 ng/ml recombinant mouse TNF-α (410-MT/CF, R&D Systems) for 4 or 8 h and harvested for cell adhesion assay or Western blot.