Epiquik nuclear extraction kit 2

Protein was isolated from treated cells (as previously described) using EpiQuik Nuclear Extraction Kit II (Epigentek, Farmingdale, NY, USA) following the manufacturer's directions. DNMT inhibition was analyzed using EpiQuik DNA Methyltransferase Activity/Inhibition Assay Kit (P-3001-2, Epigentek) following the manufacturer's directions.

LSD1 inhibitor Tranylcypromine 1/2H₂SO₄ (TCP, Merck, 13492-01-8) was dissolved in ddH2O. Concentration of TCP was 40.0 μM based on our previous study and inhibition curve we obtained. The medium was replaced with fresh medium containing the drug daily. Nuclear extract/LSD1 were prepared using the Epi-Quik™ Nuclear Extraction Kit II (Epigentek, OP-0022-100) and LSD1 activity were tested by the EpiQuik™ Histone Demethylase LSD1 Activity/Inhibition Assay Kit (Epigentek, P-3079-96) following the manufacturer’s instructions, respectively.

For the detection of Nrf2 and PGC‐1α, cytosolic and nuclear fractions were prepared using the EpiQuik Nuclear Extraction Kit II (Epigentek, Farmingdale, NY, USA). SH‐SY5Y cells and PGC‐1α over‐expressing cells (~7 × 10⁶ cells) were harvested by trypsinisation. For the detection of TFEB, nuclear and cytoplasmic fractions were prepared from ~2 × 10⁶ cells by scraping in to a hypotonic buffer (10 mM Hepes (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.15% NP‐40), and homogenising with 20 strokes of a Dounce homogenizer. An aliquot (100 μL; 25% total) was reserved for total lysate and was supplemented with 1% (w/v) SDS and 10 units of DNase (Promega, Madison, WI, USA). Remainder was centrifuged at 17 000 g for 5 min at 4°C. Supernatant (cytosol) was removed and the nuclear pellet resuspended in 200 μL high salt buffer (20 mM Hepes, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5% (v/v) NP‐40) and solubilised with SDS (1% (w/w) final and 10 U DNase (Roczniak‐Ferguson et al. 2012). Protein concentration of each fraction was measured using bicinchoninic acid protein assay kit.