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- supplemental material - Supplemental Figure 1. The effects of NMDA and KCl on arginine uptake are reversible. Treatment with 50 ?M NMDA or 40 mM KCl reduces arginine uptake in primary cortical neurons (1.86 + 0.22 for control; 1.14 + 0.15 for 50 ?M NMDA; 1.13 + 0.16 for 40 mM KCl in pmol/well). The effects of NMDA and KCl are readily reversible, as the arginine uptake is fully restored after removal of NMDA or KCl from media (2.17 + 0.31 for control; 2.15 + 0.23 for 50 ?M NMDA; 1.92 + 0.23 for 40 mM KCl in pmol/well).
- supplemental material - Supplemental 2. (A) Effect of KCl treatment on arginine uptake. Forty mM of KCl reduces the arginine uptake, which is not fully blocked either by 20 ?M of NMDA receptor antagonist APV, or by 1 ?M of sodium channel blocker tetrodotoxin, or 2 ?M of voltage-gated calcium channel (Cav1) blocker nifedipine (2.68+ 0.22 for control; 1.80 + 0.20 for 40 mM KCl; 1.75 + 0.18 for 40 mM KCl plus 20 ?M APV; 1.34 + 0.16 for 40 mM KCl plus 1 ?M TTX; 1.35 + 0.15 for 40 mM KCl plus 2 ?M nifedipine in pmol/well). (B) Activation of AMPA receptors reduces the uptake of arginine. Twenty ?M of AMPA also reduces arginine uptake. The effect is blocked by 20 ?M of AMPA receptor antagonist CNQX, but not by 20 ?M of NMDA receptor antagonist APV (2.31+ 0.18 for control; 1.34 + 0.09 for 20 ?M AMPA; 1.36 + 0.10 for 20 ?M AMPA plus 20 ?M APV; 2.08 + 0.16 for 20 ?M of AMPA plus 20 ?M CNQX in pmol/well).
- supplemental material - Supplemental 3. Effect of lentiviral-mediated RNAi of CAT1/3 on viability of primary neurons. Primary neurons infected with lentiviruses were indicated by GFP expression. Effect on cell viability following RNAi knockdown of CAT1/3 was studied by propidium iodide (PI) staining. About 60% of neurons are PI positive after 8 hour treatment with100 ?M NMDA. In contrast, only 10% of neurons are PI positive 4 days after RNAi knockdown CAT1/3, which is not different from control neurons infected with lentviruses with a scramble sequence.
- supplemental material - Supplemental 4. Effect of NMDAR blockade by APV on mTOR activity. Phosphorylation of S6 (A) and 4E-BP (B). Neurons infected with lentiviruses with scramble, CAT1, and CAT3 RNAi sequences were treated with 50 ?M NMDA or 50 ?M APV in neurobasal medium. Experiments were repeated twice with a similar result.
- supplemental material - Supplemental 5. Representative images illustrate how neurites are analyzed by Imaris software. (A). A hippocampal neuron labeled with GFP. (B). Neurites were traced with lines by Imaris software. (C). Neurites were traced proportionately to their thickness (labeled with green color). (D). Identification of spines and filopodia from branches (labeled with yellow color).
- supplemental material - Supplemental text