Hela cells

Normal primary human fibroblast TIG‐7 cells and human cervical cancer HeLa cells were purchased from the Japanese Collection of Research Bioresources (Osaka, Japan). TIG‐7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nissui, Tokyo, Japan) supplemented with 10% bovine serum (HyClone, Tokyo, Japan) on tissue culture dishes (Thermo Fisher Scientific, Waltham, MA, USA) under 5% CO₂ and 95% humidity. Similarly, HeLa cells were cultured in DMEM supplemented with 5% bovine serum, and Hrt7 cells, a HeLa cell line that expresses the reverse tetracycline transactivator, were cultured in DMEM supplemented with 7% bovine serum and 0.4% glucose 21. Cellular senescence was induced by culturing cells with MG132 (Cayman Chemical, Ann Arbor, MN, USA). The dose of MG132 was adjusted based on the cell density because a slightly higher dose of MG132 was required for the effective induction of cellular senescence when cells were plated at a high cell density to prepare protein or RNA samples: 100 nm of MG132 was used for the cells plated at a low cell density (e.g., <5 × 10³ cells/35‐mm dish), and 135 nm of it was used for those plated at a high cell density (e.g., >2 × 10⁵ cells/100‐mm dish).

HeLa cells and Hep3B cells were obtained from Japanese Collection of Research Bioresources Cell Bank and American Type Culture Collection, respectively. Cells (1.5–3 × 10⁴ cells) were cultured for a day in Dulbecco's modified Eagle medium (DMEM) containing 10% (v/v) FBS in 48-well plates. For the transfection complex, RNA (2.5 pmol) was mixed with ODAGal4 (10 pmol) at room temperature, and then Lipofectamine RNAiMAX (0.75 µg) (Thermo Fisher Scientific) was added. In some experiments, RNA (2.5 pmol) with ODAGal4 (10 pmol) was incubated at 37 °C in 10% mouse serum for 0 to 72 h, and then mixed with Lipofectamine RNAiMAX (0.75 µg). The cell culture medium was replaced with 250 µL of Opti-MEM medium (Thermo Fisher Scientific), and the RNA/ODAGal4/Lipofectamine complex was added to the cells. After 4 h, the transfection complex was removed, and the cells were cultured in DMEM with 10% FBS for 2 days before gene expression analysis. The negative control siRNA was 5′-GUACCGCACGUCAUUCGUAUC-3′ (sense) and 5′-UACGAAUGACGUGCGGUACGU-3′ (antisense).

HeLa cells were obtained from JCRB Cell Bank (JCRB9004, authenticated by STR-PCR). HCT116 cells were obtained from ATCC. HEK293T cells were obtained from Dr. Masao Seto, Aichi Cancer Center Research Institute, HFF2/T (normal human fibroblasts immortalized by telomerase) cells were established in-house^{5 (link)}. They were grown in Dulbecco’s modified Eagle’s medium supplemented with 8% fetal calf serum and antibiotics. HFF2/T/E7/KRAS G12V cells overexpressing the E7 of human papilloma virus type 16 and the activated KRAS mutant G12V were established previously^{6 (link)} and grown in the same medium.

HeLa (a human cervical carcinoma cell line) and PANC-1 (a human pancreatic adenocarcinoma cell line) cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and antibiotics. H1299 (a non-small cell lung carcinoma cell line) cells were cultured in RPMI1640 supplemented with 10% FBS and antibiotics. Three batches of Japanese human serum (#1, #2, #4) and four batches of human True A serum (#3, #5, #6, #7) were purchased from KAC Co. Ltd (Kyoto, Japan). HeLa cells were obtained from the JCRB Cell Bank (Tokyo, Japan). PANC-1 and H1299 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA).

HeLa cells were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB, Japan). Human IFNAR-deficient U5A cells and their parental 2fTGH cells were kindly provided by G. Stark (Cleveland Clinic Foundation Research Institute). The cells were maintained in a 5% CO₂ atmosphere at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Perbio Science, Switzerland) and antibiotics (Life Technologies, Carlsbad, CA). The cells were treated with an IKK inhibitor (Merck Millipore, Darmstadt, Germany) when indicated.