N ter nanoparticle sirna transfection system

Primary human monocytes were transfected using the N-TER Nanoparticle siRNA Transfection System (Sigma-Aldrich) according to the manufacturer's instructions. After initial treatment of control (DMSO) or 100 nM IκK-16 for 1 h, monocytes were transfected with miR-155 mimics or antagomirs (Life Technologies, Foster City, CA, USA) at a concentration of 40 nM/well for 16 h. Control cells were transfected with scramble miRs as a negative control (sham). After 16 h of transfection, the medium was replaced with fresh medium and the cells were stimulated with 100 ng/mL LPS for an additional 6 and 24 h. Cells were collected for RNA isolation, whereas supernatants were collected for cytokine measurements. Effective transfection was confirmed by determining miR-155 levels in isolated RNA by qRT-PCR, using RNU6B as described, ensuring upregulation of miR-155 cells transfected with mimics and downregulation of miR-155 in cells transfected with antagomirs, relative to sham conditions.

To down-regulate hOCTN1 expression, RASF were transfected with either 40 nM hOCTN1 siRNA (Sigma-Aldrich; GACAAUUUACUGUGAGUUA) or non targeting scramble siRNA (Invitrogen; nonsilencing control siRNA) using a N-TER™ Nanoparticle siRNA Transfection System^® (Sigma) according to the manufacturer’s instructions. The transfection efficiency was verified by PCR. The dependence of saracatinib biological impact on hOCTN1 expression in RASF was assessed at the molecular level by determination of c-Abl activity. RASF transfected with hOCTN1- or scramble-siRNA were incubated with 1 μM or 10 μM saracatinib and 10 ng/ml PDGF for 10 min, and total extracts were examined for c-Abl activation by Western blotting using anti-phospho Abl (Cell signaling, Danvers, USA; diluted 1:500) and GAPDH antibody (Sigma-Aldrich). The part of membrane containing GAPDH, as determined from molecular weight markers, was cut and the expression of GAPDH was detected as loading control. Western blots were quantified by ImageJ software (NIH, Bethesda, MD, USA).

NHE1 siRNA (NM_003047; SASI_Hs01_00025997) and universal negative control siRNA (SIC001) were obtained from Sigma-Aldrich. For siRNA transfection, EA.hy926 cells were grown to 50–60% confluency in gelatine-coated culture dishes and transfected with NHE1 siRNA (50 nM) or scrambled siRNA (50 nM), using the N-TER nanoparticle siRNA transfection system (Sigma-Aldrich), according to the instructions of the manufacturer. For western blotting and qPCR cells were lysed 48 h post transfection.

Small interfering RNA (siRNA) duplexes targeting VASP (5′-GGACCUACAGAGGGUGAAAdTdT-3′) were designed and synthesized by RiboBio (Guangzhou, China). The siRNA duplexes (#6267) targeting cofilin was obtained from Cell Signaling Technology. The siRNA of Gα13 (#RI12255) and RhoA (#RI14534) were obtained from Abgent (Suzhou, China). Transfection was performed using N-TER Nanoparticle siRNA Transfection System (Sigma-Aldrich) according to the manufacturer's protocol. In brief, 1×10⁴ A375 cells were plated in 35-mm dishes and cultured overnight. VASP (20 nM), Cofilin (40 nM), Gα13 (40 nM), RhoA (40 nM) and negative control siRNA (20 nM) were transfected into cells, respectively. After 72 h incubation at 37°C, the silencing efficiency was determined by Western blot using specific antibodies. After knockdown for 72 h, the effect of CuB on VASP phosphorylation and actin cytoskeleton were measured by western blot and immunofluorescence microscopy.

Human CD34 siRNA (SMARTpool: ON-TARGETplus; L-019503-00-005, sequences 5′-UAACCUCAGUUUAUGGAAA−3′, 5′-GCACUAGCCUUGCAACAUCC−3′, 5′-GCGCUUUGCUUGCUGAGUU−3′, and 5′-CCACUAAACCCUAUACAUC−3′) and non-targeting siRNA (ON-TARGETplus #1) were obtained from Dharmacon, GE Lifesciences, Buckinghamshire, UK. For transfection, C-MSC were seeded at 3.2 × 10³ cells/cm² and transfection was performed using the N-TER nanoparticle siRNA transfection system (Sigma-Aldrich), according to manufacturer’s instructions. For effective knockdown of CD34, transfection was performed twice on the same cells, with the second transfection occurring 48 h after the first. Viability measurements were taken 48 h after each transfection. RNA was collected for RT-qPCR analysis, 48 h after the second transfection.

The siRNAs targeting EZH2 (SASI_Hs01_00147882) and negative control (MISSION siRNA Universal Negative Control) were obtained from Sigma-Aldrich. Cells were transfected with 100 nmol/L final concentration of siRNA using N-TER Nanoparticle siRNA Transfection System (Sigma-Aldrich). After 9 h of incubation, the transfection medium was replaced with fresh medium containing TSA (0.1 μmol/L for NCI-H2170 and 0.5 μmol/L for ABC-1) or DMSO and the cells were incubated for an additional 24 h. After 24-h drug exposure, the cells were grown in drug-free medium for an additional 24 h, and the total RNA was isolated using RNAiso (Takara). The effect of siRNA was confirmed using SYBR green RT-PCR with THUNDERBIRD SYBR qPCR Mix (Toyobo). PCR conditions and the primer sequences are listed in Table S1.