Inhibiting Filament Formation of Non-Muscle Myosin 2A in A431 Cells

To inhibit the filament formation and function of non-muscle myosin 2 A in A431 epithelial carcinoma cells we transfected them with human S100A4 constructs. DNA constructs expressing either the wild type or an inactive mutant were prepared as previously described^{34 (link)}. The mutant form (MutS100A4), derived using the Megaprimer method⁴⁸ , lacks 13 amino acids at the C-terminal and contains a point mutation in position 81 that replaces a cysteine by serine, therefore does not inhibit NM2 assembly. Both the wild type and mutant S100A4 coding sequences were subcloned into pIRES2-eGFP plasmid (Clontech), which contains an internal ribosome entry site using restriction sites XhoI and BamHI. Using the BsaI restriction site for linearization, cells were transfected with linearized plasmids, using FuGene HD transfection reagent (Promega) according to the manufacturer’s instructions. Stable transfectants were selected with 0.4 mg/ml G418 antibiotics (Merck Millipore). After two weeks of selection in G418-supplemented medium, stably transfected cells were further selected by their GFP signal using FACSAria Cell Sorter (BD Biosciences). After selection, cells were maintained in 0.2 mg/ml G418. Six S100A4-overexpressing and five mutS100A4-overexpressing cell clones were eventually established, of which one from each group was used for aggregation and segregation studies.