Sigenome non targeting sirna 1

GeneFECTOR (3:50) (VennNova, Parkland Fl) and siRNA (40 nM final) were diluted in Opti-MEM I (Invitrogen, Paisley, UK) and equal volumes of each were mixed and incubated at r/t for 5 min. siRNA complexes were added drop wise to HUVECs cultured in Opti-MEM I medium. After incubation for 4 h, culture medium was replaced with EGM2 medium (Lonza, Wokingham, UK) overnight and subsequently by M199/10% FCS. The following validated siRNA oligonucleotides were employed^{24 (link),54 (link),61 (link)}.
HO-1 (siHO-1 seq. 2) 5′- AACAUUGCCAGUGCCACCAAG-3′ (Qiagen, Manchester, UK).
AMPKα1 siRNA (Hs_PRKAA1_5) 5′-CCCACGATATTCTGTACA CAA-3′ (Qiagen).
CREB-1 pooled sequences (Dharmacon, Epsom, UK):
5′-GAGAGAGGTCCGTCTAATG-3′; 5′-CGTACAAACATACCAGATT-3′;
5′-GAGTGGAGATGCAGCTGTA-3′; 5′-TGACTTATCTTCTGATGCA-3′.
Nrf2 pooled sequences (Dharmacon):
5′-TGACAGAAGTTGACAATTA-3′; 5′-TAAAGTGGCTGCTCAGAAT-3′
5′-CCAAAGAGCAGTTCAATGA-3′; 5′-GAGAAAGAATTGCCTGTAA-3′.
The si-GENOME Non-Targeting siRNA #1 from Dharmacon containing 4 mismatches to any human, mouse or rat gene was used as a negative control.

Prior to transfection, cells were starved for 6 h in order to achieve proper cell cycle synchronization. T98G cancer cells were transfected overnight with Dharmacon's chemically synthesized siRNA SMARTpools [human PC‐1, L‐007666‐00‐0005, ON‐TARGETplus Human PKD1 (5310) siRNA–SMARTpool, 5 nmol] and non‐targeting siRNA for control cells (D‐001210‐01‐05, siGENOME Non‐Targeting siRNA #1, 5 nmol), in dilution 1:20 in 1× siRNA buffer, using DharmaFECT 2 Transfection Reagent, 0.2 ml (Dharmacon) in dilution 1:50 in DMEM (Gibco, Thermo Fisher Scientific).18 (link), 19 (link) After 16 h, the medium was changed and the cells were treated with IgPC1 and/or HP and cultured for 24 and 48 h.

Cells were transfected with siRNA targeting human ATG5 (siGENOME Human ATG5 siRNA-SMARTpool, M-004374-04; Dharmacon) or control siRNA (siGENOME Non-Targeting siRNA #1, D-001210-01; Dharmacon) at a final concentration of 10 nM using Lipofectamine RNAi MAX (Invitrogen).