Figure 1

CD44 affiliation with lipid rafts is reduced during migration of highly-invasive breast cancer cells. Sucrose density gradient fractionation was used to isolate lipid rafts from nonmigrating (a confluent cell monolayer) versus migrating (2 hours post scratch-wounding) MDA-MB-231 cells. (A) Raft fractions were identified on the basis of peak biochemical activity of the lipid raft-affiliated enzyme alkaline phosphatase. (B) Enrichment of the marker proteins Flotillin-1 (Flot-1) and transferrin receptor (TfR) by immuno-dot blot was further used to define the identity of respectively raft versus nonraft fractions. (C) The western blot expression profile of CD44 and its binding partners in MCF-10a normal-like breast cells revealed increased recovery of raft-affiliated CD44 after 2 hours of migration compared with nonmigrating controls. This was verified by calculation of the raft affiliation ratio from the quantification of three independent experiments (histogram). The CD44 binding partners tested were exclusively recovered from nonraft fractions. (D) CD44 recovery from lipid raft fractions of highly-invasive MDA-MB-231 breast cancer cells was reduced in migrating relative to nonmigrating conditions; and was paralleled by increased CD44 recovery from nonraft fractions in migrating conditions. This observation was verified by calculation of the raft affiliation ratio from the quantification of three independent experiments (histogram). The CD44 binding partners tested were exclusively recovered from nonraft fractions. Error bars, standard error of the mean; n = 3 experiments. *P < 0.05, Student’s t test. NR, nonlipid raft; OD, optical density.