Figure 5.

Restoration of NEFH expression in MDA-MB-231 breast cancer cells slows tumor progression. (A) The cellular localization of NEFH protein in MDA-MB-231 cells transiently transfected with pcDNA3.1 and pcDNA3.1-NEFH was determined by immunofluorescence with anti-NEFH (FITC). Nuclei were visualized by staining with DAPI. The NEFH protein was not expressed in MDA-MB-231 cells transfected with pcDNA3.1 and just the nuclei was visualized in these cells. MDA-MB-231 wild type cells were also stained for NEFH to determine endogenous levels of NEFH expression and the specificity of the NEFH antibody and as NEFH protein was not expressed in MDA-MB-231 wild type cells just the nuclei was visualized in these cells. (B) Invasion and migration assays in MDA-MB-231 cells transfected with pcDNA3.1 (empty vector) or pcDNA3.1-NEFH. Twenty-four hours post transfection, cells were harvested, suspended in serum-free media and added either directly to the top chamber of transwell plates (migration assay) or to the top chamber of transwell plates that were layered with 2 mg/ml matrigel (invasion assay). Growth media containing 10% serum was added to the bottom chamber and cells were allowed to migrate or invade for 24 and 48 hours respectively. Cells attached to the membrane were fixed and subsequently stained with giemsa or crystal violet. For quantification of migrated or invaded cells X fields per well were randomly selected and counted. Data presented are the mean of 2 independent experiments § SEM. Group comparisons were carried out using Student t test, P < 0.05. (C) Representative images of the invasion assay in MDA-MB-231 cells transfected with either pcDNA3.1 (empty vector) or pcDNA3.1-NEFH. (D) Representative images of the migration assay in MDA-MB-231 cells transfected with pcDNA3.1 (empty vector) or pcDNA3.1-NEFH.