The current thinking is that keratin filaments facilitate migration of cancer cells in a manner separate from epithelial-mesenchymal transitions. All experiments were performed independently three to six occasions, using a fresh aliquot of KGN cells for each experiment. Quantitative data were analyzed by one- or two-way analysis of variance (ANOVA), followed by a Tukeys post-test for multiple comparisons; differences among means were considered statistically significant at mutation is usually etiologically significant in the development of adult GCT. The function of the mutated is not fully comprehended, however it is usually postulated to be a tumor suppressor. Overexpression of induces expression of cell death receptors of the Tumor Necrosis Factor Receptor Superfamily, particularly the cell surface death receptor, Fas (FAS, also known as TNFRSF6), which facilitates apoptosis of ovarian granulosa cells [15, 16]. In contrast, granulosa cells expressing the C134W mutant lack these death receptors and are resistant to apoptosis [16]. In spite of recent attempts to develop therapeutic approaches using genetic manipulation in mouse models [14, 17, 18] and transcriptomic analysis to identify candidate driver genes [19, 20], relatively little is known about selectively treating GCT. Current treatment of GCT entails surgical resection of the ovary and/or platinum-based chemotherapy – originally UAMC-3203 developed to specifically eliminate ovarian surface epithelial (OSE) tumors [21]. However, ovarian carcinomas, UAMC-3203 including GCT and OSE tumors, share the common trait of expressing keratin intermediate filaments. Keratin type I cytoskeletal 18 protein (K18, also known as and (Smartpool Accell and siRNA; Dharmacon RNAi, GE Healthcare, Lafayette, CO). Transfection was achieved using Lipofectamine? RNAiMAX in OptiMEM? Reduced Serum Media for final 100 nM RNAi duplexes according to the manufacturers instructions (Life Technoloies, Grand Island, NY). Cells were produced to 70?% confluency then switched to antibiotic-free DMEM/F12?+?10?% FBS before siRNA- Lipofectamine? duplexes were introduced. The cells were also exposed to Lipofectamine? and a non-targeting siRNA (siexpression was evaluated by immunofluorescence as described above. Knock-down of was also evaulated using an in-cell western assay according to the manufacturers instructions (LI-COR?, Lincoln, NE). Following a 72?h exposure to siRNA, the cells were washed with PBS, then fixed and permeabilized in 100?% MeOH. Detection of K18 and -Actin (internal control) expression was achieved using an antibody cocktail of mouse anti-human K18 (CY90; Sigma-Aldrich, St. Louis, MO) and rabbit anti-human -Actin (13E5, Cell Signaling Technolgy, Danvers, MA) followed by a secondary antibody cocktail (goat anti-mouse IgG H?+?L DyLight 800 and goat anti-rabbit IgG H?+?L DyLight 680, Cell Signaling Technology, Danvers MA). The cells were imaged using a LI-COR? Odyssey? Classic Infrared Imaging scanner. Staining intensity for K18 was normalized to the staining intensity for Rabbit polyclonal to ABCA5 -actin using the provided software. Statistical analysis All experiments were independently replicated three to six occasions, using a fresh aliquot of KGN cells (passage 23-27) to initiate each experiment. Data were analyzed by one- or two-way analysis of variance (ANOVA), UAMC-3203 followed by a Tukeys post-test for multiple comparisons; differences among means were considered statistically significant at and in KGN cells (siRNA) reduced keratin expression by 63?% as determined by in-cell western (Fig.?5a and ?andb)b) and 72?% by confocal imaging (Fig.?5c). Subsequent experiments evaluated the UAMC-3203 effect of and knockdown on FasAb-induced apoptosis in KGN cells. Treatment with Lipofectamine?, non-targeting siRNA (sisiRNA alone had no effect on caspase 3/7 activity (siRNA-treated cells compared to controls (moderately augmented the sensitivity of KGN cells to the apoptosis-inducing effects of FasAb (Fig.?6). Open in a separate windows Fig. 5 Immunodetection of K18 and -actin protein expression in KGN cells mock transfected (siand (sifollowing sitransfection. The mean ( SEM) following treatment for three impartial, replicate experiments is usually depicted. c Representative image of immunofluorescent staining of K18 and actin filaments in cultured KGN cells. Keratin (K18) filament expression stained with FITC (green); -actin filament expression stained with Phalloidin (red); nuclei stained with DAPI (blue); and merged image of FITC, Phalloidin and DAPI. Bar graph represents quantification of K18 fluorescence (mean relative light models, RLU??SEM). (and siin cultured KGN cells (or.