Genejet transfection reagent

NMuMG cells (ATCC) and NMuMG genome-modified cells^{42 (link)} were grown in DMEM containing 4.5 μg/ml glucose with pyruvate, supplemented with 10% FBS and PenStrep. Cells were seeded onto Ibidi glass bottom dishes and transfected using NMuMG-optimized GeneJet transfection reagent (SignaGen) according to the manufacturer’s instructions. 24-48 h after transfection cells were imaged in an environmental control chamber at 37 C with 5% CO₂ using a Zeiss LSM880 Airyscan microscope with a 60x oil objective NA1.4.

For the TEM experiments HeLa cells were transfected with either pNLX or pMAΔ96-120 plasmid using GeneJet transfection reagent by following the manufacturer’s protocol (SignaGen Laboratories, Rockville, MD, USA). At 24 h post transfection the cells were washed once with PBS, fixed in 2.5% glutaraldehyde/2.5% paraformaldehyde, and incubated for 1 h at room temperature then overnight at 4°C. The cells were scraped from the plates, embedded and prepared for imaging as previously described (Rodriguez-Rocha et al., 2013 (link); Wen et al., 2010 (link)). For the Gag-GFP imaging HeLa cells were seeded on glass coverslips at 50% confluency and transfected the next day with Gag-EGFP, Gag-EGFP-MAΔ96-120, or Gag-EGFP-PTAP as described above. At 18 hours post-transfection the cells were washed with PBS, fixed with 3.7% formaldehyde, and mounted onto slides using Prolong Gold anti-fade reagent with DAPI to stain nuclei (Life Technologies, Grand Island, NY). Confocal microscopy was performed in the Creighton University Microscopy core facility using a Zeiss LSM 510 META NLO confocal scanning system. Images were cropped and processed for brightness/contrast using Adobe Photoshop (Adobe Systems Inc., San Jose, CA USA).