Cell Lines for Cytoskeletal Imaging

Plasmids coding for mCherry-α-tubulin and mCardinal-ZO1-C-14 were PCR-amplified from pmCherry_α_tubulin_IRES_puro2 (kindly provided by Daniel Gerlich) [67 (link)] and from mCardinal-ZO-1-C-14 (gift from Michael Davidson; Addgene plasmid # 56179), respectively, and then ligated into pMXs-IRES-Blasticidin (Cell Biolabs Inc.). Doxycycline-inducible MDCK II cells were generated using the same two-step transduction strategy as described previously [21 (link)] and enriched by antibiotic selection using hygromycin (Life Technologies) at 1600 µg ml⁻¹ and puromycin (Sigma-Aldrich) at 2 µg ml⁻¹. Inducible MDCK II cells stably expressing mCherry-α-tubulin or mCardinal-ZO-1 were generated by retroviral transduction and subsequent selection in 5 µg ml⁻¹ blasticidine (Novus Biologicals). GFP-CEP131 was PCR-amplified from pEGFP-C2-CEP131 plasmid [68 (link)] and ligated into pRetroX-Tight-Puro using Not1 and Mlu1 cloning sites. Expression of transgenes coding for EGFP-tagged centrosomal proteins was induced by treatment of cells with 2.5 µg ml⁻¹ of Doxycycline (Sigma-Aldrich) for 24 or 48 h, as indicated.