Poly l lysine (pll)

Overall nascent proteins were examined by a EZClick™ global protein synthesis assay kit (BioVision, Waltham, MA, USA) according to the manufacturer’s instructions. N2a cells were seeded onto 96-well culture plates (1 × 10⁴ cells/well) coated with poly-l-lysine (Nacalai tesque, Kyoto, Japan). The cells were transfected with siDLG4, siFXR1, siFMR1 and siCont (1 pmol/well) and incubated at 37 °C for 24 h. The culture medium was replaced with fresh medium containing EZClick™ protein label, and cells were further incubated at 37 °C for 24 h. After incubation, the cells were fixed, permeabilized, and treated with the reaction cocktail for 30 min in the dark according to the manufacturer’s instructions. The treated cells were washed five times in the wash buffer of the kit and examined in bottom measurement mode using a Synergy H1 Multi-Mode Reader (BioTeK, Winooski, VT, USA). For data correction, the total DNA content of each well was measured according to the manufacturer’s instructions.
Cycloheximide (FUJIFILM Wako) was used as a translation inhibitor. Cells were treated with 100 µM Cycloheximide for 9 h at 37 °C in a 5% CO₂ humidified chamber.

The L4–L6 DRGs were removed under deep anesthesia with isoflurane. DRGs were digested with 5 mg/ml collagenase A (Roche Diagnostics) and 1 mg/ml dispase II (Roche Diagnostics) for 30 min at 37°C, followed by 0.05% Trypsin/EDTA (Wako) for 30 min at 37°C. Then, DRGs were dissociated in F-12 medium (Thermo Fisher Scientific) supplemented with 15% fetal bovine serum by gentle pipetting. Cells were washed with F-12 medium and were resuspended in Neurobasal medium (Thermo Fisher Scientific) supplemented with 1% B27 supplement (Thermo Fisher Scientific) and 1% GlutaMAX (Thermo Fisher Scientific). Cells were plated onto 48-well plate coated with 0.5 mg/ml poly-L-lysine (Nacalai tesque, Kyoto, Japan) and 10 µg/ml Laminin I (R&D systems, Minneapolis, MN). Control or Neat1 AAV vector was added at 5×10⁹ vector genomes (vg)/well. Forty-eight hours after transduction, the cells were treated with 5 µg/ml actinomycin D (Wako) or 100 µM 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DRB; Sigma-Aldrich Japan) to block the transcription and were collected 0, 4, 6, and 8 h after actinomycin D treatment or 8 h after DRB treatment. Quantification of mRNA levels were performed as described in the quantitative PCR method.