Lenti x cells

Embryonic stem cells (AN3-12 cells) are a clonal derivative of HMSc2, mouse embryonic stem cells derived in the laboratory (Elling et al. 2011 and 2017); NIH3T3 cells were purchased from ATCC, PlatinumE cells were purchased from Cell Biolabs, and LentiX cells were purchased from Clontech. A375 cells were obtained from Cancer Cell Line Encyclopedia. Hap1 cells were obtained from Haplogen and Hela cells from ATCC. All cell lines were confirmed to be mycoplasma negative regularly.

Lentiviruses containing validated shRnf43 (TRCN0000040790) and shPten (TRCN0000322421) were purchased from Sigma-Aldrich. The lentiviruses were produced using PEI (Polysciences, 23966) transfection, the plasmids psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259), and Lenti-X cells (Clontech). For each infection, 10.000 single cells were resuspended in 1 ml PDC medium. Polybrene was added with a final concentration of 8 μg/ml. Cell/polybrene/virus mix was centrifuged for 20 min at 800 rpm at RT. The pellet was resuspended in 100 μl PDC medium containing 5% Matrigel and plated in a Matrigel-GFR coated well of a 96-well plate. Selection started after 24 h incubation at 37°C by changing the medium to PDC medium containing 5% Matrigel supplemented with 3 μg/ml puromycin.

pLKO.1-shSCR, -shMDM2-1, -shMDM2-2, and -shTP53 lentiviruses were produced by reverse transfection of Lenti-X cells (Clontech) as described (17 (link)) except using 17.5 μg pLKO.1 vector, 4.38 μg pVSV-G, 8.75 μg pMDL, 4.38 μg pREV, and 105 µl 0.6 μg/μl polyethylenimine per 15 cm dish for cancer cell lines and 5 μg pVSV-G, 10 μg pMDL, 5 μg pREV, and 160 µl 0.6 μg/μl polyethylenimine per dish for cone precursors. Supernatants were collected at 48 h post-transfection and filtered through 0.45 μm filter and used directly or concentrated by centrifugation (17 (link)). shMDM2-1, shMDM2-2, and shSCR infections used 5 ml unconcentrated virus for 5 × 10⁵ cells. For co-knockdown, 100 μl concentrated shTP53 supernatant was combined with 5 ml unconcentrated supernatant before infections, with 4 μg/ml polybrene (Sigma-Aldrich) and pipetted 20 times. After 4–6 h, virus-infected cells were diluted with 8 ml medium. Infected cells were selected starting 48–50 h post-infection with 1–2 μg/ml puromycin for up to 48 h for cancer cell lines or 2.5 μg/ml puromycin for 28 h for cone precursors. BN- and BN-MYCN transduction and selection of SH-SY5Y cells was as described (17 (link)). BE-GFP-MYCN and BE-GFP-MYCC lentivirus were produced, concentrated, and used to transduce developing retina as described (21 (link)).