Amaxa huvec nucleofector kit

HUVECs were transfected with a pool of 3 target-specific 20–25 nt TSP-1 siRNAs (Santa Cruz Biotechnology, #SC-36665) designed to knock-down gene expression. As a control, cells were treated with an equal amount of nonspecific control RNA (Dharmacon, Thermo Fisher Scientific). Transfection of synthetic RNA (200 nM siRNA or control) was done using Amaxa™ HUVEC Nucleofector™ kit according to the manufacturer's protocol (Lonza). Following transfection with siRNA, cells were incubated for 24 h followed by the conditions indicated.

Human umbilical vein endothelial cells (HUVECs; from Cascade Biologics/Invitrogen) were cultured (passages 1 to 4) on 0.2% gelatin-coated tissue culture dishes (Corning), and maintained in M200 medium (Invitrogen) supplemented with low serum growth supplement LSGS (Gibco; Cascades Biologics; Invitrogen), 2% fetal bovine serum, 1 μg/ml hydrocortisone, 10 ng/ml human epidermal growth factor, 3 ng/ml basic fibroblast growth factor, 10 μg/ml heparin, and 50 μg/ml gentamycin (Wisent). Downregulation of endogenous CdGAP expression was achieved by electroporating between 5 × 10⁵ and 5 × 10⁶ HUVECs at passage 4 with 20 nM of either non-targeting No2 siRNAs (Dharmacon) or CdGAP siRNAs from Ambion (GGAGUCACCUCAAACAUACtt sense, GUAUGUUUGAGGUGACUCCtg anti-sense)29 (link) using the Nucleofector II (program U- 001; Lonza) and the Amaxa HUVEC Nucleofector Kit following the manufacturer’s recommendations. Forty-eight hours after nucleofection, cells were either serum-starved before VEGF stimulation and lysed, or processed for the various in vitro biological assays.

siRNAs or plasmids were transfected into cells by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) or the Amaxa HUVEC Nucleofector kit (Lonza Group Ltd., Basel, Switzerland) according to the manufacturers' instructions. RNA inhibition sequences were synthesized in GenePharma (Shanghai, China). The siRNA sequences targeting NEAT1, NEAT1-2, TLR3, TLR4, SFPQ, NONO, and IRF7 were described previously (11 (link), 44 (link)). Notably, si-NEAT1 targets both NEAT1-1 and NEAT1-2, while the stealth RNA NEAT1-2 (st-NEAT1-2) is specific for NEAT1-2. The sequences of nontarget control (i.e., negative control [NC]) or targeted RIG-I and DDX60 were designed by GenePharma as follows: NC, 5′-UUCUUCGAACGUGUCACGUTT-3′; si-RIG-I-1, 5′-GCCCAUUUAAACCAAGAAATT-3′, si-RIG-I-2, 5′-GGUGGAGGAUAUUUGAACUTT-3′, and si-RIG-I-3, 5′-CCCAACGAUAUCAUUUCUTT-3′; si-DDX60-1, 5′-GUCCAGGUGUCAGUUUGAUTT-3′, si-DDX60-2, 5′-CCGAAGUGAAGAAGGUAAATT-3′, and si-DDX60-3, 5′-GAUGGAUGCUAGGAAAUAUTT-3′.
The pCMV-NEAT1-1 and pCMV-NEAT1-2 plasmids, which transcribe NEAT1-1 and NEAT1-2, respectively, were provided by Nakagawa Shinichi (10 (link)). The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen.

HUVEC were transfected with siRNA against syndecans4 (S12639, Ambion, Carlsbad, CA, USA) using a nucleofector apparatus (Amaxa, Basel, Switzerland) and the Amaxa HUVEC Nucleofector kit (Lonza, Basel, Switzerland) following the manufacturer’s instructions. In total, 5 × 100,000 cells were resuspended in 100 µL HUVEC Nucleofector solution and transfected with 40 nM siRNA against syndecans4 and Silencer Negative Control siRNA #1 (AM4611, Ambion, Carlsbad, CA, USA). Syndecans4 silencing efficiency was determined by qRT-PCR. Silenced cells were treated 24 h after the transfection.