Single Cell Migration Assay Using Human Breast Cancer MDA-MB-231 Cell Line.

Cell migration is a fundamental cellular process that plays a crucial role in many physioglogical and pathological processes such as wound healing or cancer metastasis. Many assays have been developed to examine cell migration, such as the wound healing or scratch assay, Boyden Chamber or transwell assay, and the method we will describe here, single cell migration assay. In this assay, cells are plated sparsely on a collagen coated plate and live cell imaging is performed over a period of 2 h at 1 frame per minute. After imaging is completed, cells are tracked manually using ImageJ by tracking movement of the centroid of the cell. These data points are then exported and overall distance travelled from frame to frame is determined and divided by total time imaged to determine speed of the cell. This method provides a quick way to examine effect of cellular manipulation on cell migration before proceeding to perform more complex assays.

An in vitro Assay to Screen for Substrates of PKA Using Phospho-motif-specific Antibodies.

Kinases function as regulators of many cellular processes such as cell migration. These enzymes typically phosphorylate target motif sequences. Mass spec or phospho-specific antibody detection can be used to determine whether a kinase can phosphorylate proteins of interest, however, mass spec can be expensive and phospho-antibodies for the protein of interest may not exist. In this protocol, we will describe an in vitro kinase assay to provide a preliminary readout on whether a protein of interest may be phosphorylated by PKA. Our protein of interest is purified after expression in bacteria and treated with recombinant PKA from bovine heart. Protein is then extracted and a western blot is performed using a phospho-specific antibody for PKA's target motif. This will allow us to quickly determine if it is possible for PKA to phosphorylate our protein of interest.

Effect of oxidized cellulose on human respiratory mucosa and submucosa and its implications for endoscopic skull-base approaches.

BACKGROUND: Regenerated oxidized cellulose (ROC) sheets have gained popularity as an adjunct to a vascularized nasoseptal flap for closure of dural defects after endoscopic endonasal skull-base approaches (EESBS). However, evidence supporting its impact on the healing process is uncertain. This study was performed to evaluate the impact of ROC on the nasal mucosa and assess its effects on tissue pH, structure, and cell viability. METHODS: In 5 patients, a 1-cm2 piece of ROC gauze was placed on the surface of the middle turbinate before it was resected as part of a standard EESBS. Mucosa treated with ROC was separated from untreated mucosa and a histologic examination of structural changes in the respiratory epithelium was performed. To assess the effect of ROC on pH, increasing amounts of ROC were added to culture medium. Nasal fibroblasts viability was assessed in the presence of ROC before and after the pH was neutralized. RESULTS: Compared with unexposed controls, treated mucosa exhibited a higher incidence of cell necrosis and epithelial cell detachment. When added to Dulbecco's modified Eagle medium, ROC caused a dose-dependent decrease in pH of the medium. Only 1 ± 0.8% of cultured fibroblasts exposed to the ROC-induced acidic medium were alive, whereas 98.25 ± 0.5% of the cells were viable when the pH was neutralized (p < 0.001). CONCLUSION: ROC applied in vivo to nasal mucosa induced epithelial necrosis likely by diminishing the medium pH, because pH neutralization prevents its effect. The ultimate effect of this material on the healing process is yet to be determined.

BRCA1 deficiency in ovarian cancer is associated with alteration in expression of several key regulators of cell motility - A proteomics study.

Functional loss of expression of breast cancer susceptibility gene 1(BRCA1) has been implicated in genomic instability and cancer progression. There is emerging evidence that BRCA1 gene product (BRCA1) also plays a role in cancer cell migration. We performed a quantitative proteomics study of EOC patient tumor tissues and identified changes in expression of several key regulators of actin cytoskeleton/cell adhesion and cell migration (CAPN1, 14-3-3, CAPG, PFN1, SPTBN1, CFN1) associated with loss of BRCA1 function. Gene expression analyses demonstrate that several of these proteomic hits are differentially expressed between early and advanced stage EOC thus suggesting clinical relevance of these proteins to disease progression. By immunohistochemistry of ovarian tumors with BRCA1(+/+) and BRCA1(null) status, we further verified our proteomic-based finding of elevated PFN1 expression associated with BRCA1 deficiency. Finally, we established a causal link between PFN1 and BRCA1-induced changes in cell migration thus uncovering a novel mechanistic basis for BRCA1-dependent regulation of ovarian cancer cell migration. Overall, findings of this study open up multiple avenues by which BRCA1 can potentially regulate migration and metastatic phenotype of EOC cells.