Laminin 1

We coated 96-well plates overnight at 4 °C with recombinant Npnt (5 μg/ml, R&D Systems), collagen I (10 μg/ml, Nippi), collagen IV (10 μg/ml, Nippi), laminin I (10 μg/ml, Nippi), and fibronectin (10 μg/ml, Nippi), then they were washed with PBS and blocked with 3% bovine serum albumin for 1 hour at 37 °C. M3H1 cells were then plated at a concentration of 2 × 10⁴ cells/well and cultured for 48 hours. Sox2+ cells were determined by immunohistochemistry using an anti-Sox2 antibody. Total cells were counted using DAPI, then the ratio of Sox2+ cells was determined under a fluorescence microscope.

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.