4.1. Cell Culture

TIG-3 and RPE-1 cells were obtained from Japanese Cancer Research Resources Bank (JCRB, Osaka, Japan) and mouse primary fibroblasts (MEFs) were established from day 13.5 mouse embryos. Wild type (WT) and atg5-/- immortalized MEFs [48] were obtained from RIKEN Cell Bank (RCB2710 and RCB2711, respectively) (RIKEN BRC, Ibaraki, Japan). These cells were cultured in Dulbecco’s Modified Eagle’s (DME) medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS). THP-1 cells were obtained from Cell Resource Center for Biomedical Research, Tohoku University. They were cultured in RPMI 1620 medium (Nacalai Tesque) supplemented with 10% FBS. In this research, young control cells were less than 40 population doublings and replicative senescent cells were more than 70 population doublings. TIG-3 cells were infected with recombinant retroviruses encoding Ras^V12 (in pBabe–puro) or SMS2 or nSMase2 (in pMarX–puro) cDNA and treated with puromycin for 7 days [22]. For senescence induction, cells were irradiated with 10 Gy or 20 Gy X-rays using CP-160 (Faxitron X-ray Inc., Arizona, USA). An nSMase inhibitor GW4869 (Sigma-Aldrich, St. Louis, MO, USA), spiroepoxide (Santa Cruz, Dallas, TX, USA), doxorubicin (Wako, Osaka, Japan), chloroquine (Sigma-Aldrich), bafilomycin A1 (Sigma-Aldrich), rapamycin (Calbiochem, Merck, Darmstadt, Germany), PMA (Promega, Madison, WI, USA) or 3-MA (Calbiochem) were used in some experiments. Bacillus Calmette-Guérin (BCG) was purchased from Japan BCG Laboratory (Japan BCG Laboratory, Tokyo, Japan). All culture cells were confirmed the absence of mycoplasma contamination.