Pspax2

EVs isolated from thirty ml of conditioned medium were collected from cells cultured at 70% confluency in two 100 mm plates after 72 h (seeding density 2.2 × 10⁶ cells/plate). The conditioned media was centrifuged at 300 × g for 10 min to remove intact cells, dead cells, and cell debris. The medium was then concentrated using a centrifugal concentrator with a 100,000 molecular-weight cutoff (Amicon^®Ultra-15 Centrifugal filters), yielding about 0.5 ml concentrate (two spins of 10 ml at 6000 × g for 10 min). This concentrate was resolved by passing through IZON qEV original size exclusion columns (SEC) followed by 15 ml of double filtered (0.2 μm) PBS. Five-hundred-microliter fractions were collected. High particle/low protein fractions (from 7 to 11) were pooled and concentrated using Amicon^®Ultra-0.5 Centrifugal filters to a final volume of 200 µL at 10,000 × g for 30 min. The typical yield of an EV isolation was approximately 7.1 × 10⁷ ± 3.2 × 10⁷ particles/ml. This method was adapted to isolate EVs, LVVs (transgene plasmid, psPAX2 (Addgene #12260) and pMD2.G (#12259), VSV G-VLPs (pMD2.G (Addgene #12259), and GAG-VLPs (psPAX2 (Addgene #12260)) before being exposed to scProteins (see below). LVVs purified from media of 2.5 million HEK293Tcells transfected with psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) were isolated with SEC.

Lentivirus expressing shRNA was produced by co-transfecting HEK293T cells with a mixture of shZNF384 or shControl plasmid, the Gag-Pol packaging plasmid psPAX2 (Addgene, cat#12260), and the envelope plasmid pMD2G (Addgene, cat#12259) at a 4:3:2 ratio using Lipofectamine 2000 transfection (lip2000) reagent (Thermo Fisher, cat#11668019). Lentivirus expressing sgRNA and cas9 was produced by co-transfecting HEK293T cells with a cocktail of gRNA/Cas9-expressing lentiCRISPRv2, Gag-Pol packaging plasmids, including psPAX2 (Addgene, cat#12260), and B19/VSVG (Addgene, cat#88865) at a ratio of 3:4:1 using lip2000 reagent. Virus particles were harvested 48 h after transfection. Lentivirus-infected cells were screened with the corresponding concentration of puromycin (Thermo Fisher, cat#A1113802: Huh7, 1 µg/µl; PLC/PRF/5, 1 µg/µl; HCCLM3, 10 µg/µl).

Lentivirus stocks were produced as previously described with slight modifications. Human embryonic kidney 293FT cells (Invitrogen) were transfected using Lipofectamine 2000 (Thermo Fisher) with the expression of two helper plasmids: psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) Ten micrograms of the transfer vector, 5 µg psPAX2 and 5 µg pMD2.G of DNA were used per 10‐cm plate. Forty‐eight hours after transfection, the supernatants of four plates were pooled, centrifuged at 780 × g for 5 min, filtered through a 0.45‐µm pour size filter, and further centrifuged at 24 000 rpm for 2 h. The resulting pellet was re‐suspended in 100 µL of PBS. Lentivirus titration was performed on DIV5‐6 at titer range 10⁷ IU/mL. In most experiments, cultures were infected overnight, rinsed twice with virus‐free medium the next morning and incubated in normal culturing medium for another 48–72 h prior assay.