Fig. 4

The bromodomain inhibitor PFI-1 increases sense RNA foci in C9BAC primary cortical neurons. a Representative confocal images (upper row) of fixed 10 DIV cortical neurons derived from C9BAC (C9BAC) and non-transgenic (Ntg) mice analyzed by RNA FISH using a 5′Cy3-tagged G2C4 DNA probe (red) targeting the sense C9ORF72 repeat transcripts. Samples were also stained with DAPI (blue) to visualize the nucleus (blue). Black-and-white inverted images (lower row) are depicted to optimize visualization of the RNA foci. Brightfield microscopy was used to identify neurons from glial cells (not shown). Images show the presence of sense RNA foci in C9BAC (but not in Ntg) cortical neurons, that are increased in number after treatment with PFI-1 (5 µM in 0.05% DMSO) for 24 h. No RNA foci are detected after RNAse treatment. b Quantification of RNA foci in the different conditions as indicated. Evaluated nuclei were separated in populations of > 10 or ≤ 10 sense RNA foci. Bars show the mean percentage ± S.E.M. of each population with respect to total quantified nuclei in each condition. ***P < 0.001 relative to untreated C9BAC cells, one-way ANOVA (n = 3 independent experiments with a minimum of 30 nuclei quantified for each condition)