FIGURE 2.

Expanded CGG repeats within the FMR1 5′UTR form intranuclear RNA aggregates and FMRpolyG protein aggregates in granulosa cell lines. HGrC1 cells were transfected with a plasmid expressing either 100 CGG repeats within the FMR1 5′UTR or no CGG repeats (control) and analyzed 24 h after transfection by RNA FISH using a (CCG)8x‐Cy3 DNA probe counterstained with DAPI or fluorescence microscopy for the presence of CGG RNA aggregates (A) or FMRpolyG protein (B), respectively. (C) Western blotting using an FMRpolyG‐specific antibody ³⁹ following transfection of Δ5′UTR FMR1 (CGG)100x GFP and 5′UTR FMR1 (CGG)100x GFP plasmids confirms only the latter is capable of producing FMRpolyG protein, with a band (green) visible at ~37–40 kDA, corresponding to FMRpolyG itself and a GFP tag. ACTB (red) was used as a loading control. (D) HGrC1 cells were transfected with a plasmid expressing either 60x CGG repeats, 100 CGG repeats within the FMR1 5′UTR or no CGG repeats (control) and analyzed at 24, 48 and 72 h after transfection by RNA FISH. Whilst RNA aggregates formed following expression of 60x CGG repeats increased in size and number over time, RNA aggregates formed following expression of the 5′UTR FMR1 (CGG)100x GFP plasmid were stable in size and number. (E) Immunostaining for p62 expression in CGG‐RNA aggregate‐positive and FMRpolyG‐positive cells. Scale bars represent 10 μM.