Establishment of MDA-MB-231 and A549 cell clones overexpressing ESRP1. (A) The expression of panCD44, CD44v9 and ESRP1 was determined by western blot analysis (asterisk, CD44s; arrow, CD44v8-10). Two ESRP1-overexpressing clones (clones 1 and 2) of each cell line were examined in subsequent experiments, and compared with empty vector mock-transduced cells. (B) Messenger RNA expression of CD44 was determined by reverse transcription-polymerase chain reaction using two sets of primers. Primer set 1 detected all isoforms of CD44, while primer set 2 specifically detected CD44v8-10 and CD44v10. (C) Flow cytometric analysis of the total amount of CD44 protein was determined using an anti-panCD44 antibody. WB, western blot; CD, cluster of differentiation; CD44v, variant isoform of CD44; ESRP1, epithelial splicing regulatory protein 1; EV, empty vector; Cl, clone; CD44s, standard isoform of CD44; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PE, phycoerythrin; Ig, immunoglobulin.