2.6.3. Histone extraction, Western blot analysis and protein extraction

After stimulation for 48 h with the compounds at 10 μM and 50 μM, ORY-1001 (Selleck Catalog No. S7795), commercially available LSD1 inhibitor, was used as positive controls. ORY-1001 was used at the final concentration of 25 μM. Cells were collected and washed 2 times with PBS then processed for histone extraction. Pellets were resuspended in triton extraction buffer [TEB; PBS containing 0.5% Triton X 100 (v/v), 2 mmol/L PMSF, 0.02% (w/v) NaN₃], and the lysis was performed for 10 min at 4 °C. The samples were centrifuged at 2000× g for 10 min at 4 °C and pellets were washed in TEB (half volume). Samples were then resuspended in 0.2 N HCl, and acid histone extraction was carried out overnight at 4 °C. The supernatants were recovered, and protein concentration was quantified by Bradford assay (Bio-Rad). For each sample, 4 μg of proteins were loaded on 15% polyacrylamide gels. The nitrocellulose filters were stained with Ponceau red (Sigma-Aldrich, Schnellendorf, Germany) as an additional control for equal loading. H3K4me2, H3K9me2 (Diagenode, Ougrée, Belgium; pAB-035–050, pAb-060–050), and H4 (Cell Signalling #2592) were used according to the manufacturer’s instructions.