L-Glutamate, phorbol 12-myristate 12-acetate (TPA), valproic acid (VPA), and dihydrokainic acid (DHK) were all obtained from Tocris Biosciences (Ellisville, IL, USA). 5-Aza-2’-deoxycytidine (A3656), D-Aspartic acid (A8881), Bisindolylmaleimide I (203290) and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-YY1 (c-20) (sc-281), anti-TET1 (4F4) (sc-293186), anti-Laminin a/c (sc20681) were obtained from Santa Cruz Biotech (Santa Cruz, CA, USA), anti-DNMT3b (ab2851) and anti-EAAT1/GLAST (ab416) were purchased from Abcam Inc. (Cambridge, UK), anti-TET1 (GTX627420), anti-GADPH (GTX239) and anti-Tubulin (GTX114) were purchased from GeneTex (Irvine, CA, USA). The monoclonal anti-actin antibody was a generous gift from Prof. Manuel Hernández (Cinvestav, Mexico City). Horseradish peroxidase-linked secondary antibodies and the enhanced chemiluminescence reagent (ECL) as well as tissue culture reagents were obtained from GE Healthcare (Carlsbad, CA, USA) and from Gibco by Life Technologies (Carlsbad, CA, USA).
Primary cultures of cerebellar BGC were prepared from 14-day- old chick embryos (Avimex, Mexico City, Mexico) as previously described and characterized . Cerebella were dissected, cut into small pieces and incubated for 15 min at 37 °C in Puck’s medium containing trypsin (0.25 mg/mL) and DNase (0.08 mg/mL) to dissociate the tissue. The media was removed and substituted with Opti-MEM containing 2.5% fetal bovine serum (FBS), 2 mM glutamine, and gentamicin (50 μg/mL) for mechanical dissociation. BGC were then recovered by the repeated removal of dissociated cells and diluted to 1 × 106 cells/mL and then were seeded in plastic culture dishes. The cultures were maintained at 37°C under standard conditions (5% CO2 and 95% humidity) and used on the 4th–7th day after culture.
The MIO-M1 cell line was grown as previously described . The cells were subcultured by detachment with Trypsin-EDTA 10X (5.0 g/L trypsin, 2.0 g/L EDTA and 8.5 g/L NaCl). After removal of culture medium, 0.05% Trypsin-EDTA was added, and the cells were incubated at 37 °C during 2-3 min until cells were detached without losing adherent characteristics. The reaction was stopped by addition of 10% fetal bovine serum (FBS) in DMEM. The cells were detached by pipetting, collected in a 15mL conic tube, centrifuged at low speed and the supernatant was discharged, the cell pellet was resuspended in DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. Trypan-stained and live cells were counted by optical microscopy in a Neubauer chamber and viability was expressed as percentage of live cells with reference to the total number of cells. Cells were then seeded in 6-well plates at a density of 1 × 105 cells/well, p60 plates at 1x109 cells/plate or in 24-well plates at 250 000-500 000 cells/well and incubated at 37 °C until reaching the desire confluence.
SDS-PAGE and Western blots
Confluent monolayers were harvested with phosphate-buffer saline (PBS) (10 mM K2HPO4/KH2PO4, 150 mM NaCl, pH 7.4) containing phosphatase inhibitors (10 mM NaF, 1 mM Na2MoO4 and 1 mM Na3VO4). Total protein extraction was performed with RIPA buffer (50 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, 1mM phenylmethylsulfonylfluoride, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1% NP-40, 0.25% sodium deoxycholate, 10 mM NaF, 1 mM Na2MoO4 and 1 mM Na3VO4, pH 7.4). Cytoplasmic protein extraction was performed with buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF and 50 μg/μl protein inhibitors cocktail) followed by nuclear protein extraction using buffer C (20 mM HEPES, pH 7.9, 0.4 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF and 40 μg/μl protein inhibitors cocktail). Cell lysates were denaturized in Laemmli's sample buffer, and equal amount of proteins (approximately 70 μg as determined by the Bradford method) were resolved through 10% or gradient (6-12%) SDS-PAGE slab gels and then electroblotted to nitrocellulose membranes. Blots were stained with Ponceau S stain to confirm that protein content was equal in all lanes. Membranes were soaked in PBS to remove the Ponceau S and incubated in TBS containing 5% dried skimmed milk and 0.1% Tween 20 for 2 h to block the excess of non-specific protein binding sites. Membranes were then incubated overnight at 4°C with primary antibodies as indicated in each figure, followed by the respective secondary antibodies incubation for 2h at room temperature. Immuno-reactive polypeptides were detected by chemiluminescence with a MicroChemi (DNR Bio-imaging System) charge-coupled device (CCD) imager (DNR; MahaleHaHamisha, Jerusalem, Israel). Densitometry analyses were performed with Image J64 application (NIH; Bethesda, Maryland, USA) and data analyzed with Prism 5, GraphPad Software (San Diego, CA, USA).
Genomic DNA isolation and DNA Methylation Quantification
Genomic DNA (gDNA) was extracted using the Wizard Genomic DNA Purification Kit (Promega; Madison, WI, USA) according to the instructions provided by the manufacturer. Quality and concentration of samples was evaluated using an Epoch Spectrophotometer (Bio-Tek, USA), and through its integrity analysis in 1.5 % agarose gels. The DNA Colorimetric Quantification Kit (ab117128) (Abcam, Cambridge, UK) was used to determine global DNA methylation of glial cells according to the manufacturer’s instructions. Briefly, binding buffer was added to each well then, a negative control, a positive control, or 100 ng of gDNA per reaction were added. The plate was incubated for 90 min, washed, and incubated with the capture antibody for 60 min. The plate was washed and incubated with the detection antibody after which an enhancer solution was added. Finally, the plate was washed and developing solution was added followed by stop solution. Absorbance was determined at 450 nm. The relative methylation status was calculated according to manufacturer’s protocol.
RNA extraction and RT-qPCR assay
Total RNA was extracted with TRIzol reagent (Invitrogen; Carlsbad, CA, USA) according to the manufacturer’s protocol. The RNA concentrations were determined by spectrophotometry with Nano-Drop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The mRNA levels were analyzed using the KAPA SYBR FAST One-Step (KapaBiosystem; Boston, MA, USA) according to manufacturer’s protocol. The conditions of reverse transcription and amplification were 37 °C during 30 s, 42 °C during 5 min and 95 °C during 5 min, followed by 40 cycles of amplification: 5 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C; melt curve: 15 s at 95 °C, 1min at 60 °C and 15 s at 95 °C. Reactions were performed in StepOnePlus Real-Time PCR System (Applied Biosystems; Foster City, California, USA). Data was normalized with GAPDH as an internal control and the relative expression were calculated using the 2-(ΔΔCt) method. Primer sequences were: DNMT1 forward GGTTCTTCCTCCTGGAGAATG, DNMT1 reverse GTCTGGGCCACGCCGTACTG; DNMT3A forward GGTGCTGTCTCTCTTTGATG, DNMT3A reverse ATGCTTCTGTGTGACGCTG; DNMT3B forward ACCACCTGCTGAATTACTCACG, DNMT3B reverse GATGGCATCAATCATCACTGG; and GAPDH forward CCGGGAAACTGTGGCGTGATGG, GAPDH reverse AGGTGGAGGAGTGGGTGTCGCTGTT.
D-Aspartate uptake was carried out as previously described . Cultured monolayers in 24-well dishes were treated under different stimulus (indicated in the figures) for the indicated time periods. All the treatments were made in complete medium. Once the incubation periods finished, cultured monolayers were washed twice with pre-warmed uptake buffer (HEPES-buffered solution containing 25 mM HEPES, 130 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 33.3 mM glucose, and 1 mM NaH2PO4, pH 7.4), and were incubated with fresh pre-warmed uptake buffer containing 0.4 µCi/mL [3H]-D-aspartate ([3H]-D-Asp) (specific activity: 16.5 Ci/mmol, Perkin Elmer, MA, USA). D-Asp (glutamate analogue) was used as it has the advantage of being non-metabolizable while still is taken up by the same transporters systems as Glu. Uptake was finished after 30 min of incubation and monolayers were washed with ice-cold uptake buffer; cells were lysed with 0.1 N NaOH. Protein concentration in the lysates was determined with Bradford protein assay (Bio-Rad, CA, USA) and then were transferred to scintillation vials. Radioactivity was measured in a PerkinElmer Tri-Carb 2810TR liquid scintillation counter (PerkinElmer, MA, USA). Experiments were performed in quadruplicates in three–four independent cultures.
All data was first evaluated by the Shapiro-Wilk test to determine normal distribution. If the data was normally distributed a one-way ANOVA test vas used for statistical analysis. If the data were not normally distributed, the non-parametric Kruskal-Wallis test was performed. When these analyses indicated significance a Dunnett’s, Bonferroni’s or Dunn´s post hoc test was used. The Prism 5 software (GraphPad Software, La Jolla, California, USA) was used for all the statistics, all the results are presented as mean ± standard deviation (SD). In all cases, a p-value less than 0.05 was considered statistically significant.