Tubacin sml0065

The transformed and metastasising fibroblasts used here were previously constructed by Hahn et al. by the insertion of three well-defined genetic elements: the telomerase catalytic subunit (hTERT) in combination with two oncogenes (the simian virus 40 large-T oncoprotein; an oncogenic allele of H-ras, V12) into neonatal Bj human dermal fibroblasts [23 (link)]. These provided the control Bj primary cells, immortalised Bjhtert cells, cell cycle-defective BjhtertSV40T cells, and the final BjhtertSV40TRasV12 variant that forms tumours that metastasise to lungs in immunodeficient mice. These cell variants can be considered as an isogenically matched model system of the transformation of primary cells to metastasising cells, and they have been characterized previously with regard to their total gene transcription and protein expression, as well as the cytoskeletal organisation and behaviour of the cells [18 (link),25 (link),40 (link)]. The cells were cultured as described previously [40 (link)]. BjhTertSV40TRasV12 fibroblasts were treated with 10 μM tubacin (SML0065; SigmaAldrich, St. Louis, MO, United States) or dimethylsulphoxide and incubated at 37 °C for 4 h.

The cells were seeded at 1500 cells/well in slide chambers (543079; Greiner Bio-One Frickenhausen, Germany) and left to grow for 4 days in Dulbecco’s modified Eagle’s medium supplemented with 10% foetal bovine serum and 100 U/mL penicillin and 100 microg/mL streptomycin. Just prior to live-cell imaging, the medium was replaced with fresh medium containing 0.05 μg/mL Hoechst (H3570; Invitrogen, Waltham, MA, United States) with 10 μM tubacin (SML0065; Sigma Aldrich, St. Louis, MO, United States) or dimethylsulphoxide (BP231-100; Thermo Fisher Scientific, Waltham, MA, United States). This was followed by 24 h live-cell imaging under a microscope (Cell Discoverer 7; Zeiss, Oberkochen, Germany), with images taken every 45 min.