3 deazaneplanocin a dznep

The human NSCLC cell lines 95C, 95D and YTMLC‐90, provided by Professor Zhou from Shanghai Pulmonary Hospital, Shanghai, China, were used for experiments. 95C and 95D are human giant‐cell lung cancer cell lines with low and high metastatic activity, respectively, from the same patient. YTMLC‐90 is a lung squamous cell line. These cells were cultured in RPMI 1640 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% FBS (Gibco BRL) in a humidified atmosphere of 5% CO₂ at 37°C. We purchased 3‐deazaneplanocin A (DZNep) and tranylcypromine (2PCPA) from Selleck Chemicals LLC (Houston, TX, USA).

2 x 10⁴ or 1 x 10⁵ cells were plated in 24-well or 12-well plates, respectively, and counted at the indicated time points with or without treatment. EZH2 inhibitor GSK343 was used at 2 µM concentration throughout the manuscript unless otherwise indicated. 3-deazaneplanocin A (DZNeP) was purchased from Selleckchem (S7120). Cell numbers and viability were measured using Guava ViaCount assay on Guava easyCyte Flow Cytometer system (Millipore) and analyzed with the Guava CytoSoft software package. Cell-cycle was analyzed by BrdU/PI staining and detection of apoptotic cells in xenograft-derived tumor sections was analyzed by TUNEL staining 35 (link). SA-β-gal staining was used to detect senescent cells in vitro and in vivo. Both chromogenic and flow cytometry-based fluorescent assays were used in this work. Detailed protocol was reported previously 36 (link).

PPC cell line LNCaP was purchased from American Type Culture Collection. Mycoplasma testing was performed using a Mycoplasma Detection kit (Beijing Solarbio Science & Technology Co., Ltd.). Bicalutamide^®-resistant cells (Bica-R) and Enzalutamide^®-resistant cells (Enza-R) were developed by treating LNCaP cells with Bicalutamide^® and Enzalutamide^® in our laboratory between October 2016 and February 2017 and were used as CRPC cell lines. To develop resistance, LNCaP cells were cultured with 1, 5, 10 or 25 µM bicalutamide or enzalutamide. After a month of screening, a dose of 10 µM was selected as the optimal concentration for subsequent induction of CRPC cells. All cells were cultured in DMEM/F-12 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 2 mM L-glutamine and 100 U/ml penicillin at 37°C with 5% CO₂ in a humidified incubator. The lentiviral vectors non-targeting LV-control (LV-Ctrl) and LV-short hairpin (sh)RNA targeting PLCε (shPLCε) were purchased from Shanghai GenePharma Co., Ltd. Other reagents used were as follows: DMSO (Sigma-Aldrich; Merck KGaA); AR inhibitor ARN-509 (38 nM; Medchem Express); PARP1 inhibitor AZD2281 (0.5 µM; Selleck Chemicals); AR activator DHT (10 nM; Sigma-Aldrich; Merck KGaA) and enhancer of zeste homolog 2 (EZH2) inhibitor 3-deazaneplanocin A (DZNeP; 1 µM; Selleck Chemicals).