Let 7g

For in vivo experiments, formulated C12-200 lipidoid nanoparticles (LNPs) were used to package either Control (Life Technologies Cat. #4464061) or let-7g (Life Technologies Cat. 364 #4464070-Assay ID MC11758) miRVana mimic at either 0.5 or 2 mg/kg and delivered intravenously through the tail vein. LNPs were formulated following the previously reported component ratios (Love et al., 2010 (link)) with the aid of a microfluidic rapid mixing instrument (Precision Nanosystems NanoAssemblr) and purified by dialysis in sterile PBS before injection.

3T3-L1 cells were transfected with mirVana miR-106b (LifeTechnology MC10067), let 7g-5p (LifeTechnology MC11758), 330-3p (LifeTechnology MC10732), 19b-5p (LifeTechnology MC13042), 142-3p (LifeTechnology M C10398),125a-5p (LifeTechnology MC12561), 331-3p (LifeTechnology MC10881) mimics or miR-106b or Let7g antagomirs (AM10067, MH11758 respectively). 72 h after transfection, 3T3-L1 cells were evaluated for 2-DG uptake and mRNA expression in lysates by RT-qPCR. 3T3-L1 cells were also transfected with pre-miR-106b siRNA sense oligonucleotides 5’-CCU AAU GAC CCU CAA GCC GUU-3 and antisense 5’-CGG CUU GAG GGU CAU UAG GUU-3’ then exposed to VD( + ) HSC or VD(-) HSC-recipient peritoneal macrophage media for 72 h before assessment of pre-miR-106-5p or mature miR-106b-5p. Peritoneal macrophages from VD( + ) HSC or VD(-) HSC recipients were transduced with lentivirus containing sense either Jarid2-siRNA (LifeTechnology 4390771), Ppargc1a-siRNA (LifeTechnology, AM16708), or control-siRNA (LifeTechnology AM461) for 48 h and mRNA expression was determined as previously described^{81 (link)}.

The H2.35 cell line was directly obtained from ATCC and has been cultured for less than 6 months. The cells were authenticated by ATCC using Short Tandem Repeat (STR) DNA profiling. Cells were cultured in DMEM with 4% (vol/vol) FBS, 1x Pen/Strep (Thermo Scientific) and 200 nM Dexamethasone (Sigma). Cells were transfected with control (Life Technologies Cat. AM17010), let-7a (Life Technologies Cat. #4464084-Assay ID MH10050), or let-7g (Life Technologies Cat. #4464084-Assay ID MH11050) miRVana antiMiRs. AntiMiRs were packaged by RNAiMAX (Invitrogen) and transfected into H2.35 cells cultured in 96-well plates at a concentration of 50 nM. The number of viable cells in each well was measured at 2 and 3 days after transfection using CellTiter-Glo Luminescent Cell Viability Assay (Promega Cat. #G7570).