Superfect

The bicistronic reporter plasmid pRHF-EV71-5′UTR, which contains the EV71 IRES between Renilla and Firefly luciferase open reading frames^{13 (link)} was used as the template for synthesis of capped reporter RNA using the MAXIscript kit (ThermoFisher). SF268 cells were seeded in 24-well plates. Two hundred ng of reporter RNA, 5 µl of SuperFect (Qiagen), and 400 µl of RPMI with 10% fetal calf serum were combined and added to one well of cells. Cells were incubated at 37 °C for 4 h, medium was changed, and various concentrations of DMA-135 were added. Two days after transfection, IRES activity was determined by measuring Renilla luciferase (RLuc) and Firefly luciferase (FLuc) activities using a dual-luciferase reporter assay system (Promega).

To fractionate SV cells, adipocytes and SVP, we pooled subcutaneous (inguinal, interscapular) or visceral (gonadal and retroperitoneal) WATs. After 2 h of slow shaking at 37 °C, the suspension was spun at 800 g for 10 min; the resultant floating layer was the adipocyte layer and the pellet was a crude SV fraction. The floated adipocyte layer was collected for RNA extraction. The SV pellet was then resuspended in erythrocyte lysis buffer (0.83% NH₄Cl in H₂O) for 8 min, passed through a 70 μm mesh and then spun at 1,200 g for 5 min. The pellet was washed once in 1 × PBS, resuspended and passed through 40 μm mesh and spun at 1,200 g for 5 min. The pellet was resuspended in growth media (DMEM supplemented with 10% foetal bovine serum (FBS)). The SVP tubes that remained on the 70 μm mesh were washed off and collected in growth media and cultured on microscope coverslips that were precoated with 1% fibronectin (Sigma: item no.: F1141). SVPs were imaged 12 h later for GFP occupancy10 (link). For transfection experiments, expression vector harbouring cDNA for PDGFRβ was purchased from GE Dharmacon Openbiosystems (item no.: 30060666). Cells were reverse-transfected using Superfect (Qiagen: item no.: 301305) transfection regent following the manufacturer’s protocol. Cell media (DMEM supplemented with 10% FBS) was replaced 12 h post-transfection.

PAG protein expression was stably knocked-down in Jurkat T cells by RNA interference using Mission shRNA plasmids (TRCN0000123273, Sigma). Lentiviral particles were generated by transfecting HEK293T cells with pMD2G, psPAX2, and the shRNA plasmid using SuperFect (Qiagen). T cells were transduced by centrifugation and selected with puromycin.