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. 2017 Sep 6;37(36):8816–8829. doi: 10.1523/JNEUROSCI.2125-16.2017

Figure 7.

Figure 7.

A–D, Knockdown of Dlx2 expression by siRNA in E16.5 and E18.5 embryonic neocortical primary cultures. Primary dissociated forebrain cultures of neocortex (A, C) at E16.5 (A) and E18.5 (C) were transfected with a duplex control scrambled siRNA or two different duplex siRNAs targeting Dlx2 coding sequences. Transfection of duplex siRNA targeting Dlx2 reduced DLX2 expression (green; Ab, Ad, Cb, Cd) and concomitantly decreased GABA expression (green; Af, Ah, Cf, Ch) in embryonic neocortical primary cultures compared with control scrambled siRNA-transfected cells (Aa, Ac, Ae, Ag, Ca, Cc, Ce, Cg). Scale bars, 200 μm. E, Using quantitative RT-PCR, different versions of siRNA (siRNA constructs 1 and 2) knocked down Dlx2 expression in primary embryonic neocortical (NC) cultures, compared with a control scrambled siRNA control (p < 0.01). Controls were performed separately for each siRNA construct to Dlx2. Embryonic neocortical cells endogenously express DLX2, and were used as a positive control. Concomitantly, transfection with either Dlx2 siRNA resulted in statistically significant decreases in Gad1 and Gad2 expression (p < 0.05). F, For testing of siRNA transfection efficiency, cotransfection of both Dlx2 siRNAs and the siRNA control, and pooled Dlx2 siRNA with pcDNA3/Dlx2 plasmid was also performed in embryonic NC cells. Western analysis with anti-DLX2 antibody showed that different versions of siRNA (siRNA constructs 1 and 2) knocked down DLX2 expression compared with endogenous DLX2 levels. Coexpression of a Dlx2 expression plasmid with Dlx2 siRNA rescued DLX2 expression. An siRNA control (scrambled siRNA) and untransfected embryonic neocortical lysate were used as controls, respectively. β-Actin was used as loading control. B, D, Quantification of DLX2- and GABA-positive cells following reduction of DLX2 expression mediated by duplex siRNA in E16.5 (B) and E18.5 (D) neocortical primary cultures (n = 4). DLX2- or GABA-positive cells were counted and compared with the total of number of immunopositive cells transfected with scrambled siRNA control. All comparisons were performed in at least three trials. Average ± SEM with associated p values.