y treating with 1.0 mM staurosporine for 6 hours. APC-Annexin V and 7-AAD staining was performed according to the manufacturer’s instructions. Each group contains three isotype controls: unstained cells; cells stained with APC Annexin V alone; cells stained with 7-AAD alone. Samples were analyzed by flow cytometry. PARP Activity Assay PARP activity was measured using HT PARP in vivo Pharmacodynamic Assay II kit. After treatment with 100 mM carboplatin PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19672638 for 
45 minutes, A2780/CP70 cells transfected with negative control or shRNA-ALDH1A1 were washed twice with 5 ml of warm PBS. Cells were scraped into 300 ml of cold cell lysis buffer and incubated on ice for 15 minutes. SDS was added to samples to a final SDS concentration of 1% and incubated at 100uC for 5 minutes. When samples cooled to room temperature, 0.01 volume of 100X Magnesium cation and 2 ml of DNase I were added to the samples and incubated for additional 90 minutes at 37uC. After a brief centrifugation, supernatants were collected and polyADP-ribose levels PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19673813 in the cell extracts were quantified using ELISA method. Clinical Correlation All the participants provided their written informed consent to participate in this study and the institutional review board at the University of South Alabama Health System approved this study, as well as the consent procedure. Ascites from 15 consecutive patients undergoing surgery for advanced stage IIIC/IV papillary serous ovarian cancer as well as 2 patients with benign ascites were collected and was immediately processed to perform ALDEFLUOR assay after washing them with PBS and removing the erythrocytes using ACK lysing buffer. Clinicopathologic data was collected for the respective patients and correlated to percentage of cells exhibited ALDH+ phenotype in their ascites. ALDH+ ovarian cancer cells exhibits stem cell-like properties These results led us to further characterize ALDH as a potential stem-cell marker in ovarian cancer. Tumor progression can be associated with the PNU-100480 web presence of a subset of cells that express stem cell markers and exhibit aggressive behavioral properties including invasive and increased colony formation potential. To investigate their invasive properties, sorted cells were assessed using Matrigel invasion assay. As shown in the figures 3A & 3B, ALDH+ cells demonstrated over 1.7 fold increase in invasion through Matrigel compared to ALDH2 cells. Another surrogate measure to characterize cancer stem-like cells is the ability to form colonies, especially in the presence of chemotherapeutics. Consistent with their invasive potential, ALDH+ cells demonstrated enhanced colony formation ability compared with ALDH2 phenotypes. Importantly, ALDH+ phenotypes displayed enhanced colony formation ability in the presence of carboplatin were evaluated for platinum response using drug sensitivity assay as described in M&M section. To assess the percent of ALDH+ cells, in A2780 and A2780/CP70 cells ALDEFLUOR assay was performed by using BODIPY-aminoacetaldehyde as substrate for ALDH enzyme, after 40 minutes incubation at 37uC flow cytometry analysis was performed and western blot data shows representing levels of ALDH1A1 isozyme in these cells. doi:10.1371/journal.pone.0107142.g001 p,0.01). In order to gain insight into the potential stem-like mechanisms of these cells, we evaluated potential stem cell pathways of interest in these cells. The RT-PCR data showed nearly 3-fold higher level expression of Kruppel-Like Factor 4 in ALDH+ ce
