Overexpression of CD44 Isoform 12 in MB-231 Cells

MB-231 cells were transformed with the plasmid pLX304 expressing CD44 isoform 12 under the CMV promoter and a C-terminal V5 tag as previously described [33 (link),42 (link)]. In brief, 5 × 10⁴ cells were plated in each well of a 12 well plate and allowed to attach overnight in growth medium. The medium was then replaced with OptiMEM (Life Sciences, Carlsbad, CA) for 24 h. To transform cells, the medium was changed to 500 µl OptiMEM medium supplemented with 20 µl of medium containing 5 µl of Fugene (Promega, Madison, WI), 2 µg of pLX-304-CD44 isoform 12 and 98 µl of OptiMEM. Cells were allowed to grow overnight and maintained in 10 µg/ml selection drug Blasticidin S (Enzo Life Sciences, NY).