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. 2013 Aug 21;33(34):13791–13804. doi: 10.1523/JNEUROSCI.2366-13.2013

Figure 3.

Figure 3.

MEF2 does not alter synapse density or the density of synaptic proteins. A, Decreasing MEF2 activity does not change synapse density. No combination of shRNAs targeting MEF2 isoforms changes synapse density; n ≥ 63 dendrites (21 cells) per condition, two experiments. Values are normalized to shSCR control. Similarly, MEF2-En does not change synapse density from control (EGFP) levels. Cells were transfected at 6 DIV and fixed at 8 DIV. Normalized, control levels are indicated by the dashed gray line (shSCR for RNAi and EGFP for MEF2En); n ≥ 152 dendrites (51 cells) per condition, four experiments. B, Expression of MEF2-VP16 also does not alter synapse density. Cells were transfected at 6 DIV and fixed at 8 DIV; n ≥ 71 dendrites (23 cells) per condition, four experiments. C, A tamoxifen inducible form of MEF2-VP16 (MEF2-VP16-ER) does not alter synapse density. Cells were transfected with the indicated forms of MEF2 at 5 DIV. 4OHT or vehicle control (ethanol to 0.1%) was added 24 h later and cells were fixed at 8 DIV; n ≥ 21 dendrites (7 cells), two experiments. D, MEF2A+D knockdown in young cortical neurons does not alter synapse density defined by the colocalization of PSD-95 and VAMP2 (data are the same as the control shown in Fig. 7C) or by the colocalization of PSD-95 and synapsin1. Neurons were transfected at 6 DIV and fixed at 8 DIV. Normalized control (shSCR) levels are indicated by the dashed gray line; n ≥ 21 dendrites (7 cells), one experiment. E, MEF2A+MEF2D knockdown does not alter the density of PSD-95, VGluT1, synapsin1 or VAMP2. Densities of the indicated proteins were quantified from experiments shown in D, Figure 3A, and 7C. Normalized control (shSCR) levels are indicated by the dashed gray line. F, Changing MEF2 activity by dominant negative MEF2En or constitutively active MEF2-VP16 does not alter PSD95 density, but slightly decreases VGluT1. Cells were transfected at 6 DIV and fixed at 8 DIV. Normalized control (EGFP for MEF2En or MEF2-DBD-VP16 for MEF2-VP16) levels are indicated by the dashed gray line. MEF2En; n ≥ 131 dendrites (43 cells), four experiments. MEF2-VP16; n ≥ 71 dendrites (23 cells), four experiments. G, MEF2VP16 expression does not alter PSD-95 cluster area as compared with control. Cells were transfected at 6 DIV, and fixed at 8 DIV; n ≥ 71 dendrites (23 cells), four experiments. H, MEF2 shRNAs, MEF2En, and MEF2-VP16 all alter MEF2 activity as expected. shRNAs targeting MEF2A, C, or D and MEF2En decrease MEF2 activity. MEF2-VP16 increases MEF2 activity ∼3.5-fold, and a 4OH-tamoxifen inducible form of MEF2, MEF2-VP16-ER, increases MEF2 activity ∼23-fold relative to mutant versions that cannot bind DNA (MEF2-DBD-VP16 or MEF2-DBD-VP16-ER) or to MEF2-VP16-ER without 4OHT. Respective, normalized control levels are indicated by the dashed gray line; n ≥ 8 cells per condition. *p < 0.05, **p < 0.01, ***p < 0.001.