Cc117

Mouse brain vascular pericytes (iXCells Biotechnologies, 10MU-014) were grown in cell culture flasks coated in 0.01% poly-l-lysine (PLL, Trevigen, 3438-100-01). Cells were grown in Supplemented Mouse Pericyte Growth Media (SMPGM), i.e., 2% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% Pericyte Growth Supplement (iXCells, Biotechnologies, MD-0092) in an incubator at 37 °C and 5% CO₂. Pericytes were plated in PLL-coated black 96-well plates (BD Falcon, 353219) at a density of 7500 cells per well in 200 μL of SMPGM. 48 h later, the medium was replaced with SMPGM containing 0.2% FBS. For stimulation, pericytes were either treated with an inflammatory cytokine cocktail of recombinant mouse interferon gamma (IFN-γ) (PeproTech, 345-05, 10 ng/mL) and recombinant mouse IL-1β (R&D, 401-ML/CF, 10 ng/mL), or with CSPGs (Millipore, CC117, 10 μg/mL). After 48 h of treatment, the medium was discarded, and the cells were then overlaid with fresh serum-free DMEM for another 24 h to eliminate the presence of inflammatory stimuli from the culture medium. Each condition was carried out with four technical replicates. Post-24 h, the conditioned media were collected and centrifuged at 2000 rpm for 3 min to pellet any floating cells. The conditioned media were then stored at − 80 °C for analysis.

To determine the effects of CSPGs on OPC growth in vitro, flat-bottom 96-well plates were first coated with 10 μg ml⁻¹ poly-L-lysine overnight, followed by a rinsing with water. Purified CSPGs (Millipore CC-117), a commercial mixture containing neurocan, phosphacan, versican and aggrecan, were coated onto the 96-well plates at a concentration of 10 ng ml⁻¹–10 μg ml⁻¹ diluted in sterile water for 3–4 h, followed by a rinsing with water. The control wells contained poly-L-lysine alone or bovine serum albumin (1–10 μg ml⁻¹). Enriched OPCs were seeded at a density of 5.0–10.0 × 10⁴ cells per well in OPC media (n=4 wells per condition), and the plates were incubated at 37 °C and 8.5% CO₂ for 18–24 h for most experiments. The cells were fixed with 4% ice-cold paraformaldehyde at 4 °C for 10 min, rinsed with phosphate-buffered saline (PBS), and stored at 4 °C until processed for immunocytochemistry. In other experiments, OPCs were seeded onto astrocyte-secreted ECM.

Dorsal regions of neocortex were dissected from embryos at E15 or from explants 2 d in vitro (DIV) after E13 ex utero electroporation and culturing. The tissue was dissociated in 0.25% Trypsin-EDTA for 15 min in a 37°C water bath with occasional, gentle trituration. The dissociated cells were then plated in Neurobasal medium supplemented with 2% B27, 1× GlutaMAX, and 1× Pen/Strep on 24- or 96-well plates coated with 50 μg/ml poly-D-lysine (Sigma). Cultures were seeded at 1.2 × 10⁶ cells/well (24-well plate) for Western blottings, 1 × 10⁵ cells/well of a 96-well plate for subsequent microscopic analyses of morphology, or 2.5 × 10⁴ cells/well of a 96-well plate for quantitative immunocytochemistry. The cultures were maintained in a 37°C, 5% CO₂ incubator. After 2 DIV (E17 equivalent), cultures were either fixed for microscopic analyses or scraped and lysed for Western blot analyses. For exogenous CSPG exposure, purified neural CSPGs (CC117, EMD Millipore) were suspended in PBS at 50 μg/ml and added to the medium of cultured neurons at 3 μg/ml.

E18.5 pups were sacrificed and hippocampi dissected into Hank's buffered saline solution (HBSS, Gibco #14185045). The tissue was minced, trypsinised (0.025%) and triturated with heat‐polished glass pipettes. Plating was in Neurobasal medium (Thermo Fisher #21103049) with 10% FCS, 2 mM L‐glutamine (Gibco #25030149), B27 (Gibco #17504001), 10 mM HEPES (Gibco #15630056) and penicillin/streptomycin. Fifty percent of media was exchanged to FCS‐free medium after 24 h and then every 36 h. CSPG (Merck #CC117) coatings were performed as described previously (Shen et al, 2009 (link); Jin et al, 2018 (link)).

The following antibodies were used: CSPGs (2 μg/ml; CC117, EMD Millipore), cycloheximide (10 μg/ml; C0934, Sigma Aldrich), recombinant rat myelin-associated glycoprotein (MAG; 30 μg/ml; P07722, R&D Systems), Y-27632 ROCK inhibitor (10 μM; 1254, Tocris Bioscience), anti- αTAT1 (1:200; ab58742, Abcam), anti-HDAC6 (1:500; NB100-91805, Novus Biologicals), anti-acetylated α-tubulin (1:1000; D20G3, Cell Signaling Technology), anti-α-tubulin (1:5000; DM1A, Sigma-Aldrich), anti-β-actin (1:5000; AC-74, Sigma-Aldrich), anti-β III tubulin (1:5000; MRB₄₃₅P, BioLegend) and anti-GFP (1:500; Sigma-Aldrich). Lentivirus containing GFP (control) or GFP-tagged wild-type αTAT1 constructs, under the human cytomegalovirus (CMV) promoter, was purchased from Dr. Mingjie Li (Washington University School of Medicine, St. Louis, MO; Li et al., 2010 (link)). HDAC6 activity was determined using the fluorometric HDAC6 Activity Assay kit (BioVision), as per manufacturer’s instructions.