Vorinostat

Chidamide was supplied by Chipscreen Bioscience Ltd. (Shenzhen, China). ABT-199, Z-VAD-fmk, romidepsin, and vorinostat (SAHA) are all purchased from MedChemExpress (New Jersey, USA).
Molm-13 cells were purchased from AddexBio (San Diego, USA). MV4;11 and NB4 cells were purchased from ATCC (Teddington, UK). OCI-AML2, OCI-AML3 cells were kindly provided by Prof. Bing Z Carter (MD Anderson Cancer Center, USA). All cells were tested and authenticated by an AmpFlSTR Identifiler PCR Amplification Kit (Thermofisher Scientific, USA) in the year of 2018 in our laboratory, and were monthly tested for mycoplasma using PCR method. Peripheral blood samples of healthy donors for hematopoietic stem cell transplantation (n = 11) and bone marrow samples of patients with AML (n = 36) were obtained from the First Affiliated Hospital of Xiamen University with the informed consent for research purposes only. This study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Review Board of First Affiliated Hospital of Xiamen University.

T24 cells treated with 10 μM SAHA (Vorinostat; MedChem Express Co., Monmouth Junction, NJ, USA) or vehicle (DMSO) for 2 days were stained with propidium iodide and subjected to flow cytometry following standard protocols. Briefly, cells were fixed with 10 mL of 70% ethanol at –20°C overnight, stained with 0.5 mL of propidium iodide/RNase staining buffer for 15 min at room temperature, and analyzed by flow cytometry.

LMK-235(Cat# HY-18998) and Vorinostat (Cat# HY-10221) were purchased from MedChemExpress (MCE, USA). KYSE150 and TE-1 cells, treated with LMK-235 for 48 hr, were plated at 2×10³ cells/well into 96-well plate in five replicates. Cell proliferation was measured using CCK-8 kit (Dojindo, Japan) at the following day (day 0) and every 24 hr after (up to day 4). Optical density (OD) was read at 450 nm by SpectraMax M5e Microplate Reader (Molecular Devices, CA, USA).
For EdU (5-Ethynyl-2´-deoxyuridine) incorporation assay, the above-mentioned cells were seeded into glass bottom dish (35 mm) at 5×10⁴ cells/dish in triplicate. After incubation with 10 mM EdU (RiboBio Co., Ltd, China; Cat# C10310-3) for 5 hr, nuclei were counterstained with Hoechst 33342. The percentage of proliferative cells (EdU-positive) was measured within three random fields under LSM 880 confocal microscope (Carl Zeiss, Oberkochen, Germany) using 20×objective.

ACC (hTERT) cells (15,000 cells/well) were plated in 24-well plates with complete medium. For the concentration–response curves, cells were exposed to the following drugs concentration: cisplatin (Selleckchem Chemicals, Houston, Texas, USA) (0.03–24 µM); doxorubicin (Selleckchem Chemicals) (0.001–1 µM); lenvatinib (MedChem Express, Monmouth Junction, New Jersey, USA) (0.05–2 µM); vorinostat (MedChem Express) (0.1–9 µM); everolimus (Selleckchem Chemicals) (0.5–250 nM); palbociclib (Selleckchem Chemicals) (0.03–9 uM); olaparib (Selleckchem Chemicals) (0.15–18 µM). According to cells doubling time, ACC (hTERT) cells were treated with the drugs for 4 days.

Human TNBC cells (MDA-MB-231, BT-549, MDA-MB-468, HCC1806, and HCC70) were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured as per ATCC guidelines. All the model cells utilized were free of mycoplasma contamination. STR DNA profiling was used to confirm cell identity. Antibodies for GAPDH, p-S6, S6, p-Akt (S473), Akt, p-mTOR (S2448), mTOR, p-STAT3 (Y705), and STAT3 were purchased from Cell Signaling Technology (Danvers, MA). LIF and LIFR (LIFRα, CD118) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). β-actin antibody was obtained from Sigma-Aldrich (St. Louis, MO). The Ki67 antibody was purchased from Abcam (Cambridge, MA). LIFR knockout (KO) model cells and synthesis of EC359 were done using protocol in our earlier publication^{16 (link)}. Vorinostat, panobinostat, romidepsin, and givinostat were purchased from MedChemExpress (Monmouth Junction, NJ).