H3k9me2

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.

Antibodies to FLAG (catalog number F3165; Sigma), HA (clone 3F10; Roche), mCherry (catalog number ABE3523; Source Bioscience), green fluorescent protein (GFP) (catalog number ab290; Abcam), HP1α (catalog number MAB3584; Millipore), HP1γ (catalog number MAB3450; Millipore), histone H3 (catalog number ab1791; Abcam), H3K9me2 (catalog number 154-050; Diagenode), H3K9me3 (catalog number 05-1242; Millipore), H3K27me3 (catalog number 61017; Active Motif), and tubulin (catalog number 21445; Cell Signaling) were used in this study. Smchd1 rabbit polyclonal antibody was raised against a mixture of SMCHD1 fragments produced in bacteria (positions 1 to 385, 1197 to 1549, and 1615 to 1963), affinity purified, validated by Western blot analysis (see Fig. 5B), and used for experiments depicted in Fig. 5A, 7D, and 8. Smchd1 antibody (catalog number ab31865; Abcam) was used for experiments depicted in Fig. 1C and E.