Unc1999

Naïve H9 hESCs were maintained in NaïveCult expansion medium supplemented with 10 µM EED226 (Cayman Chemical, CAYM22031-10) or 2.5 µM UNC1999 (Sigma, SML0778). The latter was also applied to naïve H9 hESCs maintained in PXGL medium. Treatments lasted for 7 days, and the medium was changed daily. All cells were passaged on d3-4 counting from the start of treatment. On day 7, cells were fixed using 4% paraformaldehyde (FF-Chemicals, FFCHFF22023000) for 10 min. Samples were left in DPBS^−/− (Gibco, 14190144) at 4 °C until proceeding with the immunofluorescence protocol.

The Prestwick library (Prestwick Chemical) contains 1120 approved drugs dissolved in DMSO at a stock concentration of 10 mM. Other compounds were purchased from the following sources: from Cayman Chemical: flumequine, oxolinic acid, etoposide, mitoxantrone (hydrochloride), ellipticine, bleomycin (sulfate), hydroxyurea, actinomycin D, triptolide, and UNC1999; from Sigma-Aldrich: lomefloxacin hydrochloride, ofloxacin, and sodium salicylate; from ApexBio: amsacrine; from Santa Cruz Biotechnology: ICRF-193, sobuzoxane, and aclarubicin (aclacinomycin A); from Tocris: UNC2400. All individually purchased compounds were dissolved in DMSO or water. Chemical structures were generated using PubChem Sketcher.

Torin 2 (SML1224), CHX (C7698), insulin solution (I0516), Clomiphene citrate (C6272), ARC239 dihydrochloride (A5736), Imatinib (SML1027), UNC1999 (SML0778), SKI-II (S5696), and ISRIB (SML0843) were purchased from Sigma-Aldrich (Germany); NVP-BVU972 (S2761) and FK-506 (Tacrolimus, S5003) were obtained from Selleck Chemicals (USA); JNK Inhibitor V (420129) from Calbiochem (USA); PERK inhibitor GSK2606414 (PERKi, 516535) from Merck Millipore; Tunicamycin (11445) from Bionordika (Sweden); Thapsigargin (ab120286) from Abcam; PF-543 hydrochloride (5754) from R&D Systems (United Kingdom); ABC294640 (10587) from Cayman Chemical (USA); and the DES1 inhibitor (B-0027) from Echelon Biosciences (USA).

Seven authenticated MM cell lines were selected for UNC1999 drug response analysis^{29 (link)}. Initially, 100,000 cells/mL were seeded in flasks 24 h prior to the addition of 1 µM UNC1999 or DMSO (Sigma-Aldrich; Merck; Darmstadt, Germany; cat. no 317275). The cells received media and reagents change after 3 days. All experiments were performed after 5 days of treatment in cells originating from 3 independent cell batches per cell line.

The UM OMM1 cells were kindly provided by Dr. M. J. Jager, Leiden University Medical Center, Leiden, and Dr. Renbing Jia, Department of Ophthalmology, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine. ARPE cell line was gifted by Dr. Kang Zhang, West China Hospital, Sichuan University. Cultured explants were either exposed to GSK126 (Selleckchem, S7061), GSK503 (Selleckchem, S7804), EED226 (Selleckchem, S8496), UNC1999 (Sigma Aldrich Corp, SML0778), and EPZ6438 (Selleckchem, S7128).

Primary chondrocytes isolated from 1-week-old mice as described above were centrifuged in a 15 ml Falcon polypropylene conical tube (Corning, Tewksbury MA) for 10 min at 400 g to form chondrocyte pellets (2.4 × 10⁵ chondrocytes per pellet). Pellets were cultured in a humidified incubator at 37 °C, 5% CO₂. In the first 16 h, pellets were maintained in 500 μl regular culture medium (with 10% FBS), before switching to 500 μl culture medium with reduced FBS (DMEM/F12, 0.1% FBS, 1% Pen/Strep, 50 μg ml⁻¹ ascorbic acid) afterward. Medium was refreshed every other day. For treatment with Ezh1/2 inhibitor, DMSO or UNC1999 (2 μM final concentration, Sigma-Aldrich) was also added 16 h after pellet formation, and refreshed every other day.