Immortalized Mouse Embryonic Fibroblasts

The vim^+/+ and vim^−/− mEFs were derived from vim^+/+ and vimentin-null mice and immortalized by stable expression of SV40 large T antigen (kindly provided by J. Eriksson, Abo Akademi University, Turku, Finland). Cells were cultured in DMEM with 25 mM Hepes and sodium pyruvate (Life Technologies) supplemented with 10% FBS, 1% penicillin/streptomycin, and nonessential amino acids. All cell cultures were maintained at 37°C and 5% CO_2.For the reconstitution of vimentin protein expression in vim^−/− mEFs, the vimentin cDNA was expressed from pLVX-IRES-neo (Clonetech) by lentiviral transduction. To express the SNAP-vimentin fusion protein, the vimentin cDNA was subcloned into pLVX-IRES-puro (Clontech) with the addition of a SNAP tag (NEB). After selection of the transduced cells with the appropriate antibiotic for 1 wk vimentin expression was assessed by Western blotting using mouse anti-vimentin antibody (Sigma, clone V9, V6630) as described before (Gan et al., 2016 (link)).