Epigallocatechin gallate (egcg)

$\alpha$ -synuclein in the pT7-7 vector was expressed in Escherichia coli BL21 (DE3) and purified as previously described [50 (link)]. As a last step, $\alpha$ -synuclein was purified by size-exclusion chromatography on an ÄKTA pure chromatography system (GE Healthcare, Chicago, IL, USA) using a Superdex 200 Increase 10/300 GL (GE Healthcare) and 20 mM citric acid, pH 7, as an elution buffer. $\alpha$ -synuclein concentration was determined by measuring UV-absorption at 275 nm (extinction coefficient of 5600 M⁻¹ cm⁻¹). For the $\alpha$ -synuclein inhibition experiments, 5 mM solutions of EGCG (Tocris #4524, Bristol, UK) were prepared by dissolving EGCG in dH₂O. The EGCG solutions were frozen and stored at −20 °C, after observing no difference between freshly dissolved and thawed EGCG. EGCG_ox was prepared by dissolving 10 mM of EGCG in 20 mM citric acid, pH 7, and incubating for at least 6 h at 60 °C in a Thermomixer Compact (Eppendorf, Hamburg, Germany) at 1000 rpm. Subsequently, it was diluted to a final concentration of 5 mM, frozen, and stored at −20 °C.

To identify potential sites of epigenetic regulation of Stra8, the reference genomic sequence (NCBI Gene ID: 20899) was analyzed using the Genome Browser CpG Islands Tracks [85 (link)]. This analysis identified a region within the first exon of the Stra8 gene (chr6:34921372-34921757) meeting the following criteria of a CpG island: GC content 50% or greater (66.3%), length >200-bp (386-bp), and ratio >0.6 of observed CG dinucleotides to the expected number (0.81). For cell studies, 24 hours after plating 2.5 X 10⁴ OSCs in plastic 24-well tissue culture plates containing 0.5 ml of OSC culture medium, the cells were pretreated with 50-μM EGCG (Tocris Bioscience) for 4 hours prior to treatment with RA (2-μM) for an additional 20 hours. The culture medium was then evaluated for the number of IVD-oocytes, while adherent cells were collected in RNAzol^® RT for RNA isolation and reverse transcription with RevertAid. Quantitative PCR analysis of Stra8 mRNA levels was then performed using Stra8-specific oligonucleotide primers (forward: 3’-GAGGCCCAGCATATGTCTAAC-5’; reverse: 3’-GCTCTGGTTCCTGGTTTAATG-5’), along with primers specific for beta-2-microglobulin as a reference gene (forward: 3’-TTCTGGTGCTTGTCTCACTGA-5’; reverse: 3’-CAGTATGTTCGGCTTCCCATTC-5’), using SYBR Green (Thermo Fisher). Data were analyzed by the ΔΔC_t method of relative quantitation.

EGCG was purchased from Tocris bioscience (Cat: 4524, Minneapolis, MN, USA) and was reconstituted in dimethyl sulfoxide (DMSO) and stored at −20 °C for in vitro experiments. For in vivo experiments, EGCG was either purchased from ENZO (Cat: ALX-270–263, Farmingdale, NY, USA) or isolated from crude green tea mixture and stored at −20 °C as powder. The isolation of EGCG was performed as follows. Two (2) grams of crude green tea mixture was dissolved in MeOH/DCM 50:50 mixture and applied to a silica gel column equilibrated with a MeOH:DCM (4:96, v/v) solvent mixture. The 4–7% solvent system was used to elute EGCG and with the 7% MeOH/DCM solvent. EGCG, was collected in 16 fractions (each 25 mL). Finally, 704 mg EGCG was obtained and was of >97% purity as confirmed via NMR and LC/MS-MS analyses. Ruxolitinib was purchased from Selleckchem (Cat: S1378, Houston, TX, USA) and was reconstituted in DMSO and stored at −20 °C.