Lipofectamin rnaimax reagent

The sequence of EpCAM siRNA duplexes were 5′-UGCUCUGAGCGAGUGAGAATT-3′ and 5′-UUCUCACUCGCUCAGAGCATT-3′. Non-silencing siRNA was used as a negative control. Ovarian cancer cells were transfected with siRNAs for 72 h in the presence of Lipofectamin RNAi MAX reagent (Invitrogen, Tokyo, Japan), according to the manufacturer's protocol.

siRNA for PRMT1 (Santa Cruz Biotechnology; sc-41069) and scrambled siRNA (Quiagen, Hilden, Germany) were used for silencing the endogenous PRMT1 expression. 40 nM of each siRNA was transfected into mesangial cells using Lipofectamin™ RNAiMAX reagent (Invitrogen), following the reverse transfection method, as instructed by the manufacturers.

Murine macrophage cell line (RAW264.7) were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and Penicillin-Streptomycin (Thermo fisher Scientific, Waltham, MA, USA) in 5% carbon dioxide (CO₂) at 37°C. Then the microRNA (miR) was transfected into 1.5x10⁶/ well of macrophages in 24-well plate following a previous protocol [38 (link)]. Briefly, the transfection mixture consisted of 5 pmol of miR (miR146a, miR223 or mimic control) (Ambion, Austin, TX, USA) and 1.5 μl Lipofectamin RNAimax reagent (Invitrogen, Carlsbad, CA, USA) in Opti-MEM (Gibco, Thermo Fisher Scientific) for one reaction was incubated with cells for 2 days before the verification of transfection-efficiency by quantitative polymerase chain reaction (qPCR) as demonstrated in Fig 1. In addition, M1 or M2 polarization was activated by LPS (10 ng/ml) or IL-4 (20 ng/ml), respectively, before cell collection and supernatant cytokines were measured by ELISA (ReproTech). For preparation of adoptive transfer, miR over-expressed RAW264.7 were treated with IL-4 (20 ng/ml) to activate M2 macrophage polarization, washed 2 times by PBS and intravenously administered into mice at 1 hour prior to LPS intra-peritoneal injection (4 mg/kg).

Transient knockdown of KIF3A was achieved using small interfering RNA (siRNA) pools (siPOOLs) [49 (link)]. KIF3A siPOOLs (siKIF3A, #11127) and negative control siPOOLs (siCTRL) were obtained from siTOOLs Biotech, Martinsried, Germany. BT-12 (2.5 × 10⁵ cells/well) and CHLA-266 (5 × 10⁵ cells/well) cells were seeded on 6-well plates. The next day, transfection of siPOOLs was performed using Lipofectamin® RNAiMAX Reagent (Invitrogen, Darmstadt, Germany, #13778150) according to the manufacturer’s instructions.

NSCLC cells were plated on a 24-well plate at a density of 20,000 cells/well, grown in culture media containing 10% FBS for 24 h and exposed to triptolide (0–50 nM) for 72 h followed by methylthiazoletetrazolium (MTT, Biotium, Hayward, CA) treatment (40 μL per well) for 4 h. For assays with siRNAs, cells were transfected with HAS2, CD44 or RHAMM siRNAs (100, 200 or 50 nM, respectively, Valencia, CA) in lipofectamin RNAiMAX reagent (Invitrogen, Carlsbad, CA) for 6 h. In some experiments, A549 cells were co-treated with HA (2.5 mg/mL) and triptolide or HAS2, CD44 or RHAMM siRNAs and grown in media containing 2.5% FBS.

ICC cells (2.5 × 10⁵) in 10% FBS media were cultured in a 6-well plate for 24 h, then transfected with 15 nM siRNA targeting mRNA of MMP-9 (siMMP-9), Smad2/3 (siSmad2/3), Slug (siSlug) (Santa Cruz Biotechnology) or 15 nM AllStars siRNA Negative Control (siNeg) (Qiagen, Valencia, CA) using Lipofectamin^® RNAiMAX reagent (Invitrogen, Carlsbad, CA) following manufacturer’s protocol. After 24 or 48 h, cells were treated with or without h-TGF-β1 in 0.1% FBS media for another 24 h. Invasive ability of treated cells then was determined using the Transwell assay as described above.