Fig 1: AF1q stable overexpression induced a spindle shape cell phenotype in A2780 ovarian cancer cells(A) Western blot showing the expression of AF1q-GFP fusion protein in A2780 cells stably transfected with a GFP-tagged AF1q vector (AF1q-GFP clones A and B) as compared to cells transfected with GFP vector (mock) and untransfected cells (UNC). The blot was incubated with GAPDH antibody, as loading control. The star indicates a non-specific band. (B) Morphological appearances of mock and AF1q-GFP A2780 clones. (C) Western blot showing AF1q protein expression in A2780 cells stably transfected with a plasmid containing the full-length AF1q coding region (clones 8 and 9) as compared to cells transfected with an empty vector (mock) and untrasfected cells (UNC). As a control for loading, the blot was incubated with GAPDH antibody. (D) Morphological appearances of mock and AF1q overexpressing A2780 clones.
Fig 2: AF1q immunostaining in human ovarian serous BOTExamples of IHC staining for AF1q in BOT tumor cells (upper panel). Example of heterogeneous protein staining in BOT cells (middle panel). Negative IHC staining of AF1q in areas of BOT without evidence of atypical epithelium, which becomes detectable in the areas of transition between normal epithelium and atypical epithelial proliferation (lower panel).
Fig 3: Effect of AF1q overexpression in the onset of basal apoptosis.(A) Immunofluorescence analysis of A2780 cells transiently transfected with GFP alone (upper panels) or GFP-tagged AF1q vector (AF1q-GFP) (lower panels). After 48 hours, GFP and AF1q-GFP cells were stained with Hoechst 33342 and nuclear morphology was examined with a fluorescent microscope. Cells of interest are marked by arrows. Cells with condensed and fragmented nuclei were identified and scored as apoptotic cells. One experiment representative of three is shown. The scale bar represents 10 µm. (B) Apoptosis was represented as percentage of apoptotic cells per 100 green fluorescent cells (at least 200 cells per sample) in A2780 (left panel) and OVCAR-3 (right panel) cells transfected as in (A). Data represent the mean±S.D. of three independent experiments. Asterisks indicate significant difference (P<0.05).
Fig 4: AF1q overexpression induce acquisition of mesenchymal traits in A2780 ovarian cancer cells(A) Real-Time PCR analyses of Snai1, Snai2 and Zeb1 mRNA expression levels normalized to Gapdh mRNA level (used as an internal control) (left panel) and Western blot analysis for the expression of cytokeratins 8 and 18, vimentin, and fibronectin (right panel) in A2780 cells stably overexpressing AF1q (Cl.8 and Cl.9) compared to mock cells. As a control for loading, the blot was incubated with GAPDH antibody. (B) Growth inhibition assay in the presence of increasing concentrations of cisplatin (from 0.05 μM to 0.8 μM). (C) Real-Time PCR analyses of S100A4 mRNA levels normalized to Gapdh (used as an internal control). Asterisk indicate p-value: ** from 0.001 to 0.01 and ***< 0.001
Fig 5: Effects of 4-HPR treatment on AF1q expression in a panel of cancer cell lines.Western blot analysis for AF1q expression in OVCA432 and SKOV-3 (A) cells, in T47D and SK-N-BE (B) cells, and in OVCAR-3 and HeLa (C) cells treated for 24 hours with 4-HPR at the indicated doses. As a control for loading, the blots were incubated with actin antibody.
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