Lmk 235

For experiments 1 and 2: 30 min prior to DID, mice received IP (intraperitoneal) injections of either VEH (vehicle: 1% DMSO (Hybri-Max, Sigma Life Sciences, MO, USA) in saline (0.9% NaCl; Baxter International, IL, USA)), or 1 mg/kg CNO (1% DMSO in saline; RTI International, NC, USA). For experiment 3, 60 min prior to DID, mice were injected IP with either vehicle (5% DMSO in Dulbecco’s phosphate buffered saline; Gibco, Sigma Life Sciences, MO, USA) or 5 or 20 mg/kg LMK 235 (Selleck Chemicals, LLC, TX, USA).

Isolation of CD34⁺ cells from normal human CB samples (CordUse, Orlando, FL, USA) was performed by density gradient centrifugation over Ficoll Paque Plus (GE Healthcare, Piscataway, NJ, USA) and collection through an human CD34 MicroBead Kit (Miltenyi Biotec, San Diego, CA, USA)^{9 ,16 (link),49 (link)}. CD34⁺ cells were then cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 ng/mL stem cell factor (7466-SC-010/CF, R&D Systems, Minneapolis, MN, USA), thrombopoietin (288⁻TP-200/CF, R&D Systems), and Fms-like tyrosine kinase 3 ligand (Flt3L) (# 710802, BioLegend). For small molecule compound screen, 50,000 CD34⁺ cells were cultured in the above medium with compounds (1 μM) from the SCREEN-WELL Epigenetics Library (Enzo Life Sciences, Farmingdale, NY, USA) for 16 h. For treatment of HDAC inhibitors, the cells were incubated with M344 (S2779, 1 μM) or LMK235 (S7569, 1 μM) (Selleck Chemicals, Houston, TX, USA) for 16 h unless otherwise stated. The following compounds were also used: C646 (Selleck Chemicals, 50 μM), EML425 (Tocris Bioscience, 50 μM), Andrographolide (Tocris Bioscience, 10 μM), BMS345541 (Tocris Bioscience, 10 μM), and PDTC (Tocris Bioscience, 20 μM). Dimethyl sulfoxide (D2650, Sigma-Aldrich, St. Louis, MO, USA) was used as vehicle control.

PB (LC Laboratories, Woburn Massachusetts, USA) was reconstituted in DMSO and, for mouse experiments was diluted in vehicle (H₂O with tween 5%, PEG 5%). Specific HDAC inhibitors were reconstituted in DMSO: Romidepsin (Selleck), RGFP966 (Selleck), LMK235 (Selleck), Tasinquimod (Selleck), Tubacin (Sigma), PCI-34051 (Selleck). Recombinant Human BMP6 (R&D systems) and Recombinant Human BMP9 (R&D systems) were reconstituted in sterile 4 mM HCl containing 0.1% bovine serum albumin. LDN193189 (Sigma) was diluted in H₂O.

Cerebellar granule neurons (CGNs) were prepared from 7- to 8-day-old Sprague–Dawley rat pups as previously described (Yuan et al., 2009 (link); Wu Y. et al., 2017 (link)). Briefly, neurons were dissociated from freshly dissected cerebella and digested into single cells by trypsin in Krebs buffer. Then, 1.0 × 10⁶ cells/ml were seeded in basal medium eagle (BME) that contained 25 mM KCl and 10% fetal bovine serum. Twenty-four hours after seeding, cytosine arabinoside (10 μM) was added.
Potassium deprivation was performed as previously described (Yuan et al., 2009 (link); Wu Y. et al., 2017 (link)). Briefly, cells cultured in vitro for 7 days (DIV 7) were switched into serum-free BME medium that contained 25 mM KCl (25K) or 5 mM KCl (5K). The HDAC inhibitors SAHA, M344, VPA, and TSA and the HDAC4 inhibitor LMK235 were purchased from Selleck Chemicals (Shanghai, China).
Apoptosis rate was determined by performing nuclear staining with Hoechst 33258 (5 μM) or propidium iodide (or PI, 5 μM) as previously described (Song et al., 2006 (link); Yuan et al., 2009 (link); Wu Y. et al., 2017 (link)).

CRC cell lines (LS174T, T84, LS180, HCT15, HT29, SW620, COLO205, HCT116, COLO320, LoVo, CaCo2) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in media with supplements as described in Additional file 1: Table S2 under humidified atmosphere of 5% CO2.
Treatment with DNA methyltransferase inhibitor (DNMTi): 3.5 × 10⁴ COLO205 or SW620 cells were seeded in six-well plates and treated with 0.5% DMSO, 1.25 μM, 2.5 μM, 5 μM, and 10 μM of decitabine (Stock 50 mM in DMSO, Cat.#S1200, Selleckchem, Houston, TX, USA) for 48 h.
Treatment with histone deacetylase inhibitors (HDACi): 3.5 × 10⁴ COLO205 or SW620 cells were seeded in six-well plates and treated with 0.01% DMSO, 50 nM trichostatin A (Stock 5 mM in DMSO, Cat.# T8552, Sigma-Aldrich, St. Louis, MO, USA) and 20 nM LMK-235 (Stock 10 mM in DMSO, Cat.# S7569, Selleckchem) alone or in combination with 2.5 μM, 5 μM, and 10 μM of decitabine for 48 h.
3.5 × 10⁴ HT29, SW620, LS174T, and LoVo cells were seeded in six-well plates and treated with 8 × 10⁻³ DMSO, 5 nM, 10 nM, 20 nM, 40 nM, and 80 nM of LMK-235 for 48 h.

HeLa-AB1 cells were plated in an ibidi μ-plate 24-well plate at a density of 3 × 10⁴ cells/well. The medium was supplemented with 1 μg/mL doxycycline on day −1. On day 0, the medium was supplemented with 0.333 μM (unless otherwise specified) LMK-235 (Catalog no. S7569; SelleckChem), 0.333 μM Vorinostat (Catalog no. S1047; SelleckChem), 0.333 μM PF-06726304 (Catalog no. S8494; SelleckChem), or 1 μM of A-485 (Catalog no. S8740; SelleckChem). On day 0, the μ-plate was mounted on the 24-channel illumination apparatus located in the CO₂ incubator and illuminated for two days before flow cytometry analysis.
For Oct4-mCherry-Rex1-GFP mES cells, the 2i cocktail was removed from the mESC medium for at least four days earlier to establish heterogenous mES cell culture with Rex1-GFP-high and Rex1-GFP-low populations. Cells were then treated with 0.5 μM LMK-235 and 1 μM A-485 for two days before performing flow cytometry analysis.