Tig 1

Human cell lines, including HEK293 (embryonic kidney cells, Tet-ON 3G cell line, Clontech, Mountain View, CA, USA), HeLa (cervical cancer cells, Tet-ON 3G cell line, Clontech), HFF1 (foreskin fibroblast cells, American Type Culture Collection (ATCC, Manassas, VA, USA)), hTERT-RPE1 (hTERT-immortalized retinal pigment epithelial cells, ATCC), TIG1 (lung fibroblast cells, Japanese Collection of Research Bioresources (JCRB, Osaka, Japan) cell bank), and MRC5 (lung fibroblast cells, JCRB cell bank), were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Waltham, MA, USA) supplemented with 10% foetal bovine serum (FBS, Gibco) in a humidified incubator with 5% CO₂ at 37 ℃. For CRISPR imaging, 1 × 10⁵ cells were cultured on poly-L-lysine-coated coverslips in a 24-well plate to observe fixed cells or on a glass bottom 24-well plate for live cell imaging for 24 h. Cells were transfected with 750 ng sgRNA and 150 ng dCas9-GFP expression vectors using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA). Cells were used for imaging experiments after 48 h.
Cells were treated with 20 μM 5-Aza-dC (Sigma-Aldrich, St. Louis, MI, USA) for 72 h to induce cellular senescence, and the medium was changed daily.

The colon cancer line HCT116, the hepatocellular carcinoma lines HepG2, Hep3B, and HLE, the lung cancer lines A549 and PC9, the breast cancer lines MCF-7, and MRK-nu-1, the fibroblast cell line TIG-1, and the gastric cancer lines KATOIII and AGS were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). The breast cancer lines, SKBr3 and MDA-MB-231, and the normal mammary epithelial cell line MCF-10A, were kindly provided by H. Fujii (National Cancer Center Hospital East, Chiba, Japan). HCT116, HepG2, Hep3B, HLE, TIG-1, A549, and SKBr3 were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Sigma). KATOIII, AGS, PC9, and MDA-MB-231 were cultured in RPMI1640 (Sigma) supplemented with 10% FBS. The microvascular endothelial cells dHMVEC (Bio Whittaker, Walkersville, MD, USA) was cultured in growth factor complete endothelial basal media (EBM; Lonza, Walkersville, MD, USA) supplemented with 5% FBS. MCF-10A was cultured in MEGM BulleKit (Lonza). MCF7 was cultured in MEM (Sigma) supplemented with 0.1 mM NEAA, 1 mM sodium pyruvate, 10 μg/mL insulin, and 10% FBS. The breast cancer line MRK-nu-1 was cultured in DMEM/F12 (Sigma) supplemented with 10% FBS.

A431 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and normal human fibroblasts (WI-38, TIG-1, and IMR-90 cells) were obtained from the JCRB cell bank (Tokyo, Japan). The cultures were maintained in DMEM (Wako, Tokyo, Japan) at 37°C with 5% CO₂. Once resuscitated, the cell lines were authenticated by monitoring cell morphology.