Gsk126

AC16 human cardiomyocyte cell line was cultured in DMEM/F12 containing 2 mM L-Glutamine, 10% exosomes-free FBS, and 1% Penicillin/Streptomycin Solution. Based on our exosome uptake data, in vitro assays were performed using ExoL-10 and ExoR+3 isolated (10⁸ particles/mL) from Astronaut D14. After 24 h (post-sEV treatment), cells were co-treated with either GSK126 (1 μM, SelleckChem) for an additional 48 h, a potent EZH2 inhibitor, or paricalcitol (10 nM, SelleckChem), a synthetic vitamin D analog, for an additional 24 h (Supplementary Figure 1).

Drug concentrations were as follows except where otherwise specified: GSK-126 (5 μM, Selleckchem, S7061), vorinostat (2 μM, Selleckchem, #S1047), panobinostat (50 nM, Selleckchem, #S1030), MAK683 (5 μM, Selleckchem, S8983), docetaxel (10 nM, Selleckchem, S7787).

The activators and inhibitors used in this study were obtained from the following sources: ISO (Sigma), Forskolin (FSK, LC Laborartoy), IBMX (Adipogen), ICI (Tocris), PKI (Tocris), PRO (Tci America), GSK126 (Selleck), DZNEP (Cayman Chemical), EPZ6438 (Selleck), TSP1 peptide (Athens Research and Technology), MDV3100 (Apexbio) and Doxycycline (Enzo), TSA (Cayman). The doses and duration of their treatments were as indicated.

LNCaP-tet-AR cells were pre-cultured with 5% CSS. Cells were then plated into 12-well plates supplemented with/out 0.25 mg/ml doxycycline. After 1-2d, cells were treated with DHT for 24 hours (h) and then treated with DMSO, olaparib (S1060, Selleckchem), cisplatin (S1166, Selleckchem), or GSK126 (S7061, Selleckchem) for 0-6d. Cells were trypsinized, collected, and counted by countess II automated cell counter (Invitrogen).

Male Balb/c nude mice at the age of 4-6 weeks were obtained from Shanghai SLAC laboratory animal co. Ltd (Shanghai, China). The animal experiments were carried out in accordance with the protocols approved by the Institutional Animal Care and Use Committee of The Affiliated Hospital of Kunming Medical University. SW480 cells were transfected with SPRY4 knockdown plasmid or SPRY4 overexpression plasmid. The transfected cells were subcutaneously injected into the axilla of the mice. GSK126 (an inhibitor of EZH2, Selleck) was administrated intravenously at a dose of 150 mg/kg. The tumor volume was calculated as follows: tumor volume = 1/2 × length ×width². After 28 days, the mice were euthanized, and the tumors were weighed and collected for further tests.

THP-1 cells (Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) and monocytes were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (Gibco), 2 mM L-Glutamine, 1 mM sodium pyruvate, and 10 mM HEPES at 37°C in a humidified atmosphere containing 5% CO2.
PBMCs were isolated using Ficoll-Paque PLUS (Cytiva). Monocytes (>98% purity) were sorted from the PBMCs with a BD FACSAriaTM III cell sorter (BD Biosciences). Monocytes were identified by anti-CD14 antibody (BD Biosciences).
For transient transfection, THP-1 cells (2 × 10⁵/well) were seeded in 24-well plate 24 h before transfection. SiRNAs (combination of four different EZH2 targeting siRNAs, 200 nM) were transfected with LipofectamineTM RNAiMAX (Invitrogen). Twenty-four hours later, the cells were stimulated with 1,000 U/ml of universal IFN-I, then collected for further analysis as shown in the figures. All siRNA sequences (GenePharma) are in Table S2.
For EZH2 inhibitor treatment, THP-1 cells were pretreated with 5 uM GSK126, 5 uM Dznep (Selleck Chemicals), or DMSO for 30 min, then 1,000 U/ml of universal IFN-I was added. Then, the cells were collected for further analysis as shown in the figures.