Bix 01294

Human MDS/AML cells (SKM-1 cells) were purchased from BeNa Culture Collection (Beijing, China) and cultured in Roswell Park Memorial Institute-1640 medium containing 10% fetal bovine serum in 95% humidified air with 5% CO₂ at 37°C.
The small interfering RNA (siRNA)-NC-1, si-EZH1, si-NC-2, si-EZH2, si-NC-T2, and si-EHMT2 were designed and synthesized by GenePharma (Shanghai, China), and the sequence is shown in Supplementary Table 1. Overexpression vectors of EZH2 (pcDNA3.1-EZH2) and EHMT2 (pcDNA3.1-EHMT2) and empty vectors were constructed by Zoman Biotechnology Co., Ltd. (Beijing, China). Then, the constructed vectors and siRNAs were transfected into cells using Lipofectamine 2000 (Invitrogen Inc., Carlsbad, CA, United States).
SKM-1 cells were treated with EZH2 inhibitor EPZ-6438 (5 μM, Yeasen Biotech Co., Ltd., Shanghai, China) and EHMT2 inhibitor BIX-01294 (2.5 μM, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). Briefly, cells in logarithmic growth phase were seeded into 96-well plates (1 × 10⁵ cells/mL) supplemented with culture medium, and treated with 5 μM EPZ-6438 for 4 days (Knutson et al., 2014 (link)) and 2.5 μM BIX-01294 for 3 days, respectively (Huang et al., 2017 (link)). The treated cells were allocated as EPZ group and BIX group, respectively.

The human EC cell lines Ishikawa and HEC-1 were grown in DMEM/F12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA). All fresh cell lines were purchased from the JCRB Cell Bank (Osaka, Japan). All the cell lines used in this study were within six passages after receipt. The cell lines were not authenticated as they came from national repositories. These cell lines were routinely tested by PCR for mycoplasma contamination by using the following primers: Myco_fw1: 5′-ACACCATGGGAGCTGGTAAT-3′, Myco_rev1: 5′-CTTCATCGACTTTCAGACCCAAGGCAT-3′. The immortalized human endometrial epithelial EM cell line was generated and extensively characterized by Satoru Kyo (Shimane University, Japan) [23 (link)]. BIX-01294 was purchased from Sigma-Aldrich (St. Louis, MO). When reaching 50% confluence, HEC-1 cells were treated with different concentrations of BIX-01294 for 24 hours.

CM272 and CM579 compounds (purity >95%) was synthesized as described [21 ]. BIX-01294 was purchased from Sigma-Aldrich (purity >98%).

The HMT inhibitors used were Bix01294 (Sigma‐Aldrich, #B9311), UNC0638 (Sigma‐Aldrich, #U4885), and A‐366 (Sigma‐Aldrich, #SML4110). The HMT inhibitors were dissolved in DMSO and tested in the 1–10 micromolar range to identify concentrations that reduce Histone H3K9me3 levels with minimal toxicity (Bix01294, 2 µM, 24 hr; UNC0638, 3 µM, 24 hr; A‐366, 3 µM, 72 hr). The Zmpste24 protease inhibitor lopinavir (LPV; Cayman Chemicals, #13854) was dissolved in DMSO and used as described (20 µM, 72 hr) to inhibit proteolytic processing of prelamin A. Human fibroblasts were exposed to ionizing radiation (5 Gy), returned to the incubator for 30 min, and subsequently analyzed by IF microscopy. The siRNA to the Ran import factor NTF2 (Santa Cruz, #sc‐36105) and a control siRNA (Fisher, #AM4635) were introduced into normal human fibroblasts (80% confluence) at a concentration of 10 µM using Lipofectamine RNAiMAX (Invitrogen). The cells were analyzed ~96 hr post‐transfection.

G9a enzymatic inhibitors, BIX-01294 and UNC0638, autophagy-inhibitors, 3-methyadenine (3-MA) and chloroquine (CQ) were purchased from Sigma–Aldrich, St. Louis, MO, USA. MAPK/ERK kinase inhibitor, U0126, was purchased from Cell Signaling Technology, Danvers, MA, USA.