Pcmv dr8

For virus production, HEK-293 cells (70–80% confluency) were transfected using X-tremeGENE 9 (Roche Diagnostics Ltd., Burgess Hill, UK) according to manufacturer’s manual with 3 μg pCMV-dR 8.91 (packaging vector, Thermo Fisher) + 0.7 μg pCMV-VSV-G (envelope vector, Thermo Fisher) + 3 μg Lentiviral shRNA vectors (three pLKO1-Puro vectors targeting all Fbln2 variants of the mouse under control of the human U6 promotor (Clones: TRCN0000109479, TRCN0000109478 and TRCN0000109476 (Thermo Fisher), or a scr ctrl shRNA pLKO1-Puro control vector (Thermo Fisher)). These were mixed in 100 µl of Opti-MEM (Thermo Fisher), 6 μl X-tremeGENE 9 was added, and the transfection solution applied to the cells. After overnight incubation, the medium was replaced with fresh DMEM/10%FBS medium, and incubated for another 24 hrs, after which the virus-containing medium was collected and filtered using syringe-driven filters (Millipore Ltd., Livingstone, UK). Filtered virus was added to EpH4 cells in a 6-well plate, incubated for 4 hrs at 37 °C in a 5% CO₂ incubator, topped up with DMEM/10% FBS medium + 8 ug/ml polybrene (Sigma) and finally incubated for 24 hrs.
The medium was replaced with fresh medium containing 3 µg/ml puromycin (Sigma) for selection of transduced cells. Cells were routinely passaged with puromycin-medium to maintain the selection.

TRIM5α shRNA and plasmids, including pMD2.G, pCMVDR8.91, and pLKO-shRNA, were purchased from the National RNAi Core Facility, Genomic Research Center, Academia Sinica, Taipei, Taiwan. 293T cells (2 × 10⁵) were cotransfected with plasmids expressing TRIM5α shRNA (target sequence: 5′-CCAGACATTTGTGAATTTCAA-3′; 2.25 μg), helper plasmids pMD2.G (0.25 μg) and pCMVDR8.91 (2.25 μg), using Turbofect in vitro transfection reagent (Thermo Fisher Scientific). Culture media was changed on the following day, and after an additional 24 h, viral supernatants were collected and filtered (0.22 μM), then stored at -80°C. Plasmid pLKO-shRNA was used as a negative control. For lentivirus infection, P3HR1 cells (3 × 10⁵/mL) were transduced with the generated lentiviruses, together with 5 μg/mL of polybrene. Infected P3HR1 cells were selected using 2 μg/mL puromycin in culture medium to produce stable cell lines according to the protocol¹.