Hek293a cells

Human embryonic kidney 293 (HEK293)-A cells were purchased from Life Technologies (Carlsbad, CA, USA). The SL-CRISPR βArr1/2 KO HEK293 cell lines (βArr KO HEK293) were generated by Prof. Stephane A. Laporte (McGill University, Montréal, QC, Canada) by using the CRISPR/Cas9 method to delete β-arrestin 1 and β-arrestin 2, from a subclone of HEK293-A cells (HEK293-SL used as a control in β-arrestin experiments) [48 (link)]. Cells were maintained in DMEM medium supplemented with 10% FBS, penicillin (100 U/mL)-streptomycin (100 μg/mL)-glutamine (2 mM) at 37 °C in a humidified 5% CO₂ atmosphere.

For AAV production, Human Embryonic Kidney (HEK) 293A cells (Life Technologies) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing 4.5 g/L glucose, L-glutamine, and sodium pyruvate supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin and 1% L-glutamine (Corning Cellgro; Media Tech). For AAV neutralization assays, Chinese Hamster Ovary (CHO-K1) cells (American Type Culture Collection; ATCC) were grown in Ham’s F12K (Kaighn’s Modified) Medium (Corning Cellgro; Media Tech) supplemented with 10% FBS, 1% penicillin/streptomycin and 1% L-glutamine (Corning Cellgro; Media Tech).

Primary cortical neurons were prepared from the forebrain of ICR mice (Japan SLC) at E14.5 as described16 (link). The cortex was dissected and incubated in 0.5 ml of Ca²⁺/Mg²⁺-free Hanks' balanced salt solution with 0.05% trypsin for 5 min at 37 °C. Tissues were dissociated with 10% fetal bovine serum (FBS) in Dulbecco's modified eagle medium (DMEM) and centrifuged at 200g for 3 min. Pellets were resuspended in Neurobasal medium (Life Technologies) supplemented with 2 mM L-glutamine, kanamycin/penicillin and B-27 supplement (1:50 dilution, Life Technologies), plated at a density of ∼1 × 10⁵ cells per cm² in culture dishes, and incubated for 4 days in vitro (4 DIV) before analyses. For preparation of primary NPCs, dissociated cortical cells were cultured as floating neurospheres in DMEM/F12 (Life Technologies) supplemented with B-27 (1:50 dilution), 20 ng ml⁻¹ epidermal growth factor (PeproTech) and 20 ng ml⁻¹ basic fibroblast growth factor (PeproTech) for 48 h at 37 °C under humidified 5% CO₂ conditions. HEK293A cells (Life Technologies) and SH-SY5Y cells (gift from Dr June Biedler, Memorial Sloan-Kettering Cancer Center)26 (link) were cultured in DMEM containing 10% FBS at 37 °C under humidified 5% CO2 conditions.

Full-length Bmi1 cDNA (NCBI NM 007552.4) was synthesized from mouse E14.5 forebrain mRNA by RT-PCR. cDNAs encoding Bmi1 and its deletion mutants were subcloned into 6×Myc-pcDNA3.1+ [49] (link). Combinations of the expression vectors for necdin, 6×Myc-Bmi1, 6×Myc-Bmi1-deletion mutants, p53 and p53ΔN were transfected into HEK293A cells (Life Technologies) with calcium phosphate and harvested 24 hrs after transfection. Cell extracts (200 µg) were incubated at 4°C for 2 hrs with anti-necdin (NC243; 1∶100) and anti-Myc (9E10; 1∶4) antibodies in a lysis buffer containing 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol and 1×protease inhibitors. The complexes were pelleted with protein A-Sepharose (GE Healthcare), separated by 10% SDS-PAGE, and detected by Western blotting. For detection of endogenous necdin-Bmi1 complex, lysates of primary NPCs (500 µg protein) from E14.5 mice were incubated with rabbit anti-necdin IgG or preimmune IgG. Bound proteins were pelleted with Dynabeads protein A (Life Technologies) and detected by Western blotting as described in [49] (link).