Anti mir 155

All genes were transfected into cells using Lipofectamine RNAiMAX transfection reagent (Invitrogen)19 (link). The precursor oligonucleotide of hsa-miR-155 (pre-miR-155), the antisense oligonucleotide inhibitor of hsa-miR-155 (anti-miR-155), and their scrambled oligonucleotides were obtained from Ambion Inc. (Austin, TX, USA). To visualize the transport of artificial microRNA, we used Alexa 488-conjugated mature miR-155 as follows: 5′-Alexa488 ssH amino linker UUAAUGCUAAUCGUGAUAGGGGU-3′; complementary: 5′-ACCCCUAUCACGAUUAGCAUUAA-3′ (Gene Design, Osaka, Japan). Small interfering RNA (siRNA) targeting RAB27B and a negative control oligonucleotide were purchased from Dharmacon (Thermo Fisher Scientific, Waltham, MA, USA) to inhibit exosome secretion. RAB27B GTPases were found previously to regulate the secretion of secretory exosomes32 (link)35 (link)48 (link). Each gene and the corresponding negative control oligonucleotide were transfected.

Cells (2.5 × 10⁵) were transfected with miRNA mimics (pre-miR-155, PM12601, anti-miR-155, AM12601; Ambion) or negative control oligonucleotides using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. The final concentrations of anti-miR-155 and the miRNA mimic in the transfection mix were 250 and 50 nm, respectively. Transfection efficiency was validated by qPCR.