Mirna inhibitor

Cisplatin-resistant and sensitive cells (4 × 10⁴ cells/well) were seeded in 24 well plates. Next day OSCC cells were transfected either with 5 nM of miRNA mimic (Qiagen) or 50 nM of miRNA inhibitor (Qiagen) or mock (control) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in Opti-MEM reduced serum medium (Invitrogen Life TechnologiesInc., Carlsbad, CA) according to the manufacture’s protocol. After 6 hrs, media replaced with DMEM supplemented with antibiotics and 10% FBS and drug treatment done and incubated for 48 hrs.

DLD-1 cells were seeded in 96-well plates overnight then placed in either 20.9% or 0.2% oxygen. The next day, cells were transfected with 60 nM miRNA mimic or 120 nM miRNA inhibitor and their appropriate NTC (all from Qiagen, UK). Cells were treated with 5-FU (2 mM) the following day, for 48 h, before harvesting. Cells were fixed with 3.7% PFA for 15 min and nuclei were stained with Hoechst 33342. Cells were imaged with the IN Cell 1000 microscope (GE Healthcare, UK). Nuclei were counted using the IN Cell Developer Software v1.8.

MNCs were isolated from human whole venous blood and cord blood by conventional Ficoll-Hypaque density gradient centrifugation and resuspended in EGM-2 SingleQuots (#CC-4176, Lonza, Basel, Switzerland). Peripheral blood or cord blood MNCs (PBMNCs or CBMNCs, respectively, 4 × 10⁶ cells/mL) were seeded on a fibronectin (50 μg/mL; Sigma-Aldrich)-coated plate and incubated in a 5% CO₂ incubator at 37 °C for 4 days. After being carefully washed, adherent MNCs were transfected with or without AllStars Negative Control for siRNA and miRNA mimic experiments (#1027280, Qiagen), miScript Inhibitor Negative Control for miRNA inhibitor experiments (#1027272, Qiagen), miRNA inhibitors (80 nM, Qiagen), miRNA mimics (80 nM, Qiagen), and NF-κB p65 siRNA (80 nM, Santa Cruz) as described previously^{12 (link)–15} , followed by treatment with or without human TNF-α (10 ng/mL, R&D Systems) for another 4 days. In addition, some adherent MNCs were cultured in medium 199 supplemented with 30% human autologous serum in the presence or absence of an anti-human TNF-α antibody (5 μg/mL; #MAB210, R&D Systems) for another 4 days.

Aged cells (10⁷/sample) were nucleofected with miRNA mimics (Qiagen), miRNA inhibitors (Qiagen), negative control siRNA (Qiagen), negative control miRNA inhibitor (Qiagen) or downstream target candidate siRNAs (Origene) using the Amaxa P3 Primary Cell 4D-Nucleofector X Kit (Lonza) on a 4D Nucleofector device (Lonza), according to manufacturer’s specific protocol. Briefly, CD34⁺ cells were nucleofected with 60 nM of total miRNA mimics, miRNA inhibitors or siRNA using the “human CD34⁺ cell” program. Real time PCR for miRNA levels were similar (p > 0.05) between restored CD34+ cells and those subjected to nucleofection.