t been studied. We demonstrate that ALDH+ phenotype possesses CSC characteristics of enhanced invasion, colony formation, and stem cell markers. The specific ALDH1A1 isotype appears to be responsible for ALDH-mediated platinum resistance both clinically as well as in in-vitro models. Our data supports an ALDH1A1-mediated platinum resistance mechanism in ovarian cancer via an altered regulation of cell cycle checkpoint and DNA repair network signaling. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19672638 inhibitor, diethylaminobenzaldehyde, to serve as a negative control. Flow cytometry sorting was conducted using a BD Bioscience Aria II SORP cell sorter. Carcinogenic Properties After sorting for ALDH phenotypes, Matrigel invasion and soft agar colony formation assays were performed as previously described. To evaluate the effect of chemotherapy on colony formation, cells were treated with fresh media with 20 mM DM-1 carboplatin added every 34 days. Visible colonies were counted on five randomly selected 40X microscopic fields in each well. CellTiter-Glo Luminescent Cell Viability Assay Sorted A2780/CP70 cells were plated at density of 4000 cells per well in 96-well black plate with clear bottom. The following day, the cells were treated with various concentrations of carboplatin up to 72 hours. After 24, 48 and 72 hours, 100 ml of CellTiter-Glo reagent per well were added, incubated for 10 minutes at room temperature and luminescence was recorded on a Synergy H4 Hybrid Reader. Real-time Quantitative RT-PCR Real-time Quantitative RT-PCR was performed PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1967325 as described previously. Primers and probes for the TaqMan system were selected from the Applied Biosystems website. The relative expression mRNA levels of BMI1, c-myc, Oct3/4, KLF4, S100A1, ALDH1A1, p21, were calculated using the DDCt method and normalization to GAPDH. Lentiviral shRNA Vector Transfection Six different pGIPZ Lentiviral shRNA glycerol stocks against ALDH1A1 and one negative control shRNA were transfected according to the manufacturer’s instructions. A2780/CP70 cells were plated at a density of 26105 cells per well of a 6-well plate for 1824 hours. For each well, 2 mg shRNA plasmid DNA were transfected into A2780/CP70 using Arrest-In reagent. Puromycin was used to select the transfected cells. After optimization, ALDH1A1-knockdown efficiencies of individual shRNAs were evaluated using real-time quantitative PCR and Western Blot. Vector 398453 demonstrated the most efficient transfection with over 95% knockdown efficacy of ALDH1A1 and was used for all shRNA knockdown experiments. Materials and Methods Cell Lines and Cultures A2780 and an isogenic cisplatin resistant A2780/CP70 cell line was generated as described earlier. Western Blot Analysis Cultured cells were collected in NP-40 lysis buffer with 10 ml/ ml protease inhibitor cocktail and subjected to immunoblotting analysis by standard techniques using antibodies against ALDH1A1, FANCJ, KLF4, FANCD2, PARP-1, XRCC1, b-actin, GAPDH, and p21, phosphoChk1, BRCA1 antibodies. ALDEFLUOR Assay and Fluorescence-activated Cell Sorting To isolate the cell population with a high ALDH enzymatic activity, ALDEFLUOR assay kit was used according to the manufacturer’s instructions. After trypsinization, cells were suspended in ALDEFLUOR 
assay buffer containing ALDH enzyme substrate BODIPY-aminoacetaldehyde, and incubated at 37uC for about 40 minutes. Cells were stained using the identical conditions with the specific ALDH ALDH1A1 Maintains Stem-Like Properties by Altered DNA Repair Networks RT2 Pr
