Ntera 2

The seminoma cell line TCam-2 was kindly donated by Professor Riko Kitazawa, Ehime University Hospital [20 (link), 21 (link)]. The testicular embryonal carcinoma cell line NTERA-2 was obtained from the ATCC (Manassa, USA). All cells were free of mycoplasma contamination and used in experiments within 30 passages after thawing. TCam-2 cells were cultured in RPMI 1640 (Gibco, USA) with 10% foetal bovine Serum (FBS) (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA). NTERA-2 cells were cultured in complete DMEM (4.5 g/L glucose) (Gibco, USA) and incubated at 37 °C in a humidified atmosphere containing 5% CO₂. In vitro, cells were treated with 5–20 mM metformin (KeyGEN, China). Additionally, TCam-2 cells were treated with 5 μM cisplatin, and NTERA-2 cells were treated with 0.1 μM cisplatin (SelleckChem). The treatment strategies included monotherapy (metformin or cisplatin monotherapy for 72 h), the combination therapy (synchronous treatment with metformin and cisplatin for 72 h) and sequential treatment. Specifically, sequential treatment with metformin and cisplatin was performed by treating TCam-2 and NTERA-2 cells with metformin for 48 h and then withdrawing it for 24 h before the cisplatin exposure for another 24 h. Verteporfin (VP), which was the YAP1 inhibitor, was purchased from MedChemExpress. Both TCam-2 and NTERA-2 were respectively treated with 3 μM VP for 48 h.

Human mesenchymal stem cells (hMSCs, LONZA, PT-2501), normal human dermal fibroblasts (NHDFs, LONZA, CC-2511), HEK293T, NTERA2, HeLa cells, and induced pluripotent stem cells (iPSCs) were used in this research. hMSCs and NHDFs were maintained in Minimum Essential Medium Eagle (αMEM, MilliporeSigma, M4526) supplemented with 10% fetal bovine serum (Hyclone, SH30910.03), 1 × GlutaMAX (Gibco, Thermo Fisher Scientific, 35050–061), 1 ng/mL human basic FGF2 (Miltenyi Biotech, 130–093-840) and kanamycin (Gibco, 15160–054). Culture medium was exchanged every 2 days. FGF2 was kept at 4 °C for no longer than 1 week and was freshly added while preparing the growth medium. HEK293T, NTERA2, and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco, 11965–092) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate (Gibco, 11360–070), and kanamycin. iPSCs were induced from NHDFs as previously described [29 (link)] and maintained in StemFit AK02N (AJINOMOTO, RCAK02N). The inhibitors used in this study were LY294002 (Selleck, S1105), PD0325901 (Wako, 162–25291), and MK2206 (Selleck, S1078). All the cells were cultured in a humidified incubator with 5% CO₂ at 37 °C.