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. 2018 May 23;38(21):4985–4995. doi: 10.1523/JNEUROSCI.2370-17.2018

Figure 5.

Figure 5.

Palladin controls axonal morphology downstream of mTOR signaling. A, Hippocampal neurons were transfected with the palladin expression plasmid or control plasmid together with a GFP expression plasmid 2–6 h after plating. Five days after transfection, neurons were subjected to immunocytochemistry using the GFP (green) and Tau1 (red) antibodies. Palladin-expressing neurons had a higher percentage of neurons bearing more than one axon than control-transfected neurons. Arrowheads and asterisks indicate axons and cell bodies, respectively. B, Hippocampal neurons were transfected with the palladin expression plasmid or control plasmid together with a GFP expression plasmid, and cultured for 3 d. The neurons were subjected to immunocytochemistry using the palladin (red) and GFP (green) antibodies. The palladin intensity was significantly increased by the exogenous expression of palladin in axons. Arrowheads and asterisks indicate axons and cell bodies, respectively. C, Hippocampal neurons were transfected with the GFP-fused palladin expression plasmid or control GFP plasmid. Five days after transfection, neurons were subjected to immunocytochemistry using the GFP (green) and Tau1 (red) antibodies. Palladin-GFP-expressing neurons had a higher percentage of neurons bearing more than one axon than control-transfected neurons. Arrowheads and asterisks indicate axons and cell bodies, respectively. D, Hippocampal neurons transfected with the TSC1 RNAi or control scrambled RNAi together with the palladin RNAi 1, palladin RNAi 2, or the control plasmid were analyzed. Palladin RNAi 1 and palladin RNAi 2 target distinct regions of palladin mRNA. The neurons were subjected to immunocytochemistry using the GFP (green) and Tau1 (red) antibodies. TSC1 RNAi increased the percentage of neurons bearing multiple axons compared with control-transfected neurons, whereas palladin RNAi 1 or 2 did not alter the percentage of neurons bearing multiple axons. Palladin knockdown with both RNAi 1 and 2 significantly reduced the percentage of neurons bearing multiple axons in the background of TSC1 knockdown. E, Hippocampal neurons transfected with the TSC1 RNAi or control scrambled plasmid together with the palladin RNAi 1, palladin RNAi 2 or control scrambled plasmid were analyzed by immunocytochemistry with palladin (red) and GFP (green) antibodies. F, Hippocampal neurons transfected with the palladin RNAi 1 or control scrambled plasmid together with the SAD-A expression plasmid or control plasmid were analyzed. The neurons were subjected to immunocytochemistry using the GFP (green) and Tau1 (red) antibodies. The SAD-A expression significantly induced supernumerary axons, and the palladin RNAi1 did not suppress the supernumerary axons induced by the SAD-A expression. Scale bars, 10 μm. Asterisks indicate p < 0.05 in the all graphs.