Skip to main content
. Author manuscript; available in PMC: 2011 Jul 30.
Published in final edited form as: Biochem Biophys Res Commun. 2010 Jul 1;398(3):482–488. doi: 10.1016/j.bbrc.2010.06.104

Figure 1. Characterization of YB-1 transfected MCF-7 cells.

Figure 1

I. Electrophoretic mobility shift analyses of cytosolic (C) and nuclear (N) fractions from control (CON) and populations “A” and “B” YB-1-EGFP transfected cells. Labeled oligonucleotide comprised of the single-stranded DNA YB-1 binding sequence within the MDR1 gene promoter. Two distinct complexes were detected, denoted “<1” and “<2”. Compared to controls, extracts from population “A” cells have moderate increases in both cytosolic and nuclear YB-1, with C > N for complex 1 and N > C for complex 2. Population “B” cells, which express twice as much YB-1-EGFP protein by FACS analysis, have approximate two-fold increases in nuclear YB-1, forming complex 2. Free probe is indicated. Relative amounts of expressed YB-1-GFP were assessed by Western Blots in all cell batches used for experiments.

II. Fluorescence microscopy of control MCF-7 cells (A) expressing EGFP alone. EGFP signal is diffusely present throughout the cytosol. (B) In cells expressing YB-1-EGFP the protein is present in both, cytosolic as well as nuclear fractions, with C > N. In addition YB-1-EGFP protein is concentrated at the tips of invadopodial structures (arrow). Dual imaging of EGFP and actin (red signal) in control MCF-7 cells (C) shows no co-localization of fluorescent signals. (D) In contrast EGFP-tagged YB-1 protein co-localizes with pericellular and invadopodial actin in the transfected MCF-7 cells (yellow signal). Magnification 300 to 450-fold in A-D, magnification bars are indicated.

III. Western blot of membrane extracts for MT1-MMP protein in control (CON) and population “A” and “B” YB-1-EGFP transfected cells. As compared to controls, population “A” cells have an approximate 2-fold increase in membrane-associated MT1-MMP protein, while population “B” cells have an approximate 6-fold increase. Relative increase of MT1-MMP protein was found in all tested cell batches.

IV. YB-1-transfected MCF-7 cells have an altered distribution of DsRed2-tagged YB-1 protein. Panel A: Tagged MT1-MMP protein is symmetrically distributed in a perinuclear pattern in control MCF-7 cells. Panel B: DsRed2-tagged MT1-MMP protein is asymmetrically concentrated at the leading edge of migratory YB-1-EGFP transfected MCF-7 cells. Magnification 450-fold.