Phoenix amphotropic cells

For SV40 Large T antigen infection, amphotropic retroviruses were generated by transfecting 20 µg of pBabe-neoLargeTcDNA^{56 (link)} into Phoenix Amphotropic cells (ATCC) using calcium phosphate precipitation. Five h following transfection, fresh media was added. 24 h following transfection, retroviral supernatant was collected every 6 or 12 h for 48 h. Collected virus was filtered through a 0.45 µm filter and supplemented with 10% FBS and 4 µg/ml polybrene. Target WI38 cells were infected with retrovirus for 24 h, followed by a 24-h recovery in standard media. Cells were then selected in 600 µg/ml geneticin (Gibco) for long-term culturing.

BJ cells (ATCC) were grown in MEM, supplemented with 10% fetal bovine serum (FBS), 1% l-glutamine, 1% nonessential amino acids, and sodium pyruvate 1 mM. Phoenix amphotropic cells (ATCC) were grown in DMEM, supplemented with 10% FBS, and 1% l-glutamine. HGPS patient-derived human primary fibroblasts were grown in DMEM, supplemented with 20% FBS, and 1% l-glutamine. Informed consent had been obtained for these cells, which were donated to CNR Institute of Molecular Genetics by patient family to be used for research on HGPS. Samples belong to BioLaM biobank at CNR Institute of Molecular Genetics Unit of Bologna located in the Rizzoli Orthopedic Institute, Bologna, Italy. Normal dermal fibroblasts (NDF) harboring pTRIPZ-v5-lamin A or pTRIPZ-v5-progerin¹⁷ were grown in MEM, supplemented with 15% FBS, and 1% l-glutamine, in the presence of 1 μg ml⁻¹ puromycin. For induction of progerin expression, NDFs were cultured in the presence of doxycycline (2 μg ml⁻¹) for 4 days. All cells were grown at 37 °C, 5% CO₂.