FIGURE 5:

BRG1 knockdown down-regulates Gfap and Osmr transcription and impairs gene clustering. (A) Scheme of cell culture and virus infection procedures. NPCs isolated from E14.5 mouse telencephalon were cultured and replated on day 4. Subsequently, the cells were infected with retroviruses that expressed EGFP alone (shControl) or EGFP together with shBRG1 and then cultured with LIF (50 ng/ml) for astrocyte differentiation. (B) Quantitative RT-PCR for Gfap mRNA in shControl- or shBRG1-treated NPCs alone or stimulated with LIF for different periods of time (LIF-stim). The results were normalized to Gapdh expression and described as fold induction relative to the levels at 0 h. Each graph represents the mean (±SEM) for the relative amount of NPCs in at least three experiments. (C) Projected images of double-labeled DNA FISH in GFP-positive (green) LIF-stimulated cells for Gfap (yellow) and Osmr (red). Nuclei were counterstained with DAPI (blue). Scale bar = 5 µm. Arrowheads indicate clustering alleles. (D) Clustering frequencies determined for Gfap and Osmr in LIF-stimulated cells expressing shControl or shBRG1. Data are presented as the means ± SEM from three biological replicates (n = 73–127). Student’s t test was performed. **p < 0.01.