Transfection

Human ovarian granulosa tumour cells (COV434) were seeded for 50–70% confluence 1 day prior to transfection. For a 24 wells plate, a mixture containing 0.5 μg DNA (Extracted according to manufacture orders, using NucleoBond Xtra Midi Plus kit, Macherey-Nagel, Germany) and 1.5 μl of Mirus (MirusBio LLC, WI, USA) at a final volume of 50 μl of Dulbecco’s Minimum Essential Medium (DMEM; Biological Industries) was used. Following incubation of 30 min, the mixture was added dropwise to each well. 72 h following transfection, the cells were permeabilized in 0.5% Triton X-100 in 4% PFA for 15 min and then fixed in 4% PFA for 20 min and washed with PBSx1.

Three plasmids used for transformation were kindly provided by Dr. Charlet-Berguerand at IGBMC, France. Briefly, 5′(99CGG)-GFP contains the 5′UTR of the FMR1 gene that contains 99 CGG repeats and was fused to eGFP sequence. Poly-glycine (FMRpolyG) was fused with FLAG protein (FMRpolyG-FLAG). In ATG (99CGG)-GFP, the weak ACG start codon was replaced for a strong ATG and no expression of FMRpolyG was enabled [16].