Cell culture

The following mouse cell lines were used in this study: 3T3 spontaneously immortalized embryonic fibroblasts (ATCC), L929 spontaneously immortalized adult and adipose tissue-derived fibroblasts (ECACC), M1 myeloblast cell line (ECACC), RAW264.7 Abelson murine leukemia virus transformed macrophages (ATCC), Neuro-2a neuroblastoma cell line (ATCC) and J774A.1 reticulum cell sarcoma cell line (monocytes/macrophages) (ATCC) and were kindly provided by Thomas Michiels (de Duve Institute). Mouse embryonic fibroblasts (MEF) were prepared from CD1 mice using standard protocols and were kindly provided by Frédéric Lemaigre (de Duve Institute). ES cells were kindly provided by Olivier De Backer (Université de Namur). The human cells used in this study were previously described: HCA2 (Arnoult et al. 2010) and HFF2 (Mattiussi et al. 2012) human foreskin fibroblasts, HeLa cervix cancer cell line (ATCC), LB37 non-small cell lung cancer cell line (Tilman et al. 2009) and SW39 SV40T-immortalized fetal lung fibroblasts (Tilman et al. 2009, kindly provided by W. Wright, UT Southwestern Medical Center). Cells were cultured in EMEM (L929, Neuro-2a, SW39, HFF2, HCA2, LB37), DMEM (3T3, J774A.1, RAW264.7, MEF, HeLa), or RPMI (M1). All media were from Gibco and enriched with 1× Penicillin-Streptomycin (Gibco) and 10% FBS (Gibco). Cells were grown at 37°C under 5% CO₂. CreERT2 Terf2 F/+ and Terf2 F/F SV40T-immortalized MEF were a kind gift from Eros Lazzerini Denchi (The Scripps Research Institute) and were previously described (Okamoto et al. 2013). For RNA-IP experiments, we used 2575i immortalized mTert +/+ MEF, kindly provided by Lea Harrington (Institute for Research in Immunology and Cancer, Université de Montréal). Cells were cultured in high glucose DMEM, GlutaMAX (Gibco) supplemented with 10% tetracycline-free fetal bovine serum (Pan BioTech) and 100 U/mL penicillin-streptomycin (Gibco). For Terf2 knockout, cells were treated with 0.6 µM 4-hydroxytamoxifen (OHT, Sigma-Aldrich) and collected 48 or 72 h after treatment. Terf2 deletion was confirmed by western blotting using a rabbit monoclonal anti-Terf2 (Novus Biologicals, NB110-57130, 1:2000 dilution) and a mouse monoclonal anti-beta Actin (Abcam, ab8224, 1:5000 dilution) to control for loading. Secondary antibodies were HRP-conjugated goat anti-mouse and goat anti-rabbit IgGs (Bethyl Laboratories, A90-116P and A120-101P, 1:2000 dilution). Signal detection was performed using the ECL detection reagents (GE Healthcare) and a FluorChem HD2 imaging apparatus (Alpha Innotech).