Unc0642

Hormones and inhibitors used in culture for in vitro experiments were as follows: Glucagon (Sigma-Aldrich, catalog #G2044) was prepared in 0.05 M acetic acid (Sigma-Aldrich, catalog # 6283) at a concentration of 1 mg/ml. Forskolin (MedChem Express, catalog # HY-15371) was prepared in DMSO (Sigma-Aldrich, catalog # 2650) at a stock concentration of 10 mM. 666–15 (Sigma-Aldrich, catalog # 5383410001) was prepared in DMSO at a stock concentration of 10 mM. Palbociclib (Selleck Chemicals, catalog # S1116) was prepared in DMSO at a stock concentration of 10 mM. GSK126 (Selleck Chemicals, catalog # S7061) was prepared in DMSO at a stock concentration of 10 mM. LBH589 (Selleck Chemicals, catalog # S1030) was prepared in DMSO at an initial concentration of 10 mM and diluted to 0.1 mM for a working stock solution. Decitabine (Selleck Chemicals, catalog # S1200) was prepared in DMSO at a stock concentration of 10 mM. Sorafenib (Selleck Chemicals, catalog # S1040) was prepared in DMSO at a stock concentration of 10 mM. UNC0642 (Selleck Chemicals, catalog # S7230) was prepared in ethanol (Decon labs) at a stock concentration of 10 mM.

Olaparib (#S1060), UNC0638 (#S8071), and UNC0642 (#S7230) were obtained from SelleckChem. Full details of antibodies and usage for immunoblotting and immunofluorescence are given in Additional file 4: Table S3. Alexa Fluor 488-conjugated donkey anti-mouse secondary antibody (Invitrogen #A21202, 1:1000) was used for immunofluorescence detection of γH2AX and H3K9me2.

The therapeutic effect of WDR5 inhibitor (WDR5i) OICR-9429 (Selleckchem, Catalog No. S7833) and G9a inhibitor (G9ai) UNC0642 (Selleckchem, Catlog No. S7230) in MYCN-Amp NB cell lines IMR32, KCNR and IMR5 was determined in a checkerboard fashion. Cell lines were seeded in two 96-well plates and incubated overnight. Each combination dose had 2 replications. The next day, cell lines were treated with different dose combinations of OICR-9429 and UNC0642. Control cells were treated with DMSO. Each plate has its control cells. Cell viability was determined after 72 h using the CellTiter-Glo^® luminescent assay (Promega, catalog number G9242). Cell viability of DMSO-treated cells was set to 100%. Results were graphed with GraphPad Prism (RRID:SCR_002798). IncuCyte^® assay was used for testing the impact of synergistic effects of drug combinations on NB cell growth in realtime. Representative data from biological replicates were shown in this study. SynergyFinder (RRID:SCR_019318) online tool (22 (link)) was used to study the synergistic effect of the combination treatment of NB cells in vitro.

UNC0642 (Selleck, Houston, USA), a potent, selective inhibitor of histone methyltransferases G9a/GLP (also named EHMT1 and EHMT2), was dissolved in dimethyl sulfoxide stoke, and diluted with saline before use (dimethyl sulfoxide = 0.2% W/V in working solution). The drug was systemically administrated to the animals via intraperitoneal injection (1 mg/kg) for 3 d before fMRI scanning, and the dosage and frequency of the administration are based on a previous study [42 (link)]. For normal mice, adult animals were tested for social avoidance behavior after the final drug administration and performed fMRI scanning and tissue extraction after the behavior test. For ASS mice, the schematic timeline is shown in Fig. 5A; the baseline of social avoidance was tested on PND60, and then the stressed mice were divided into 2 groups to administrate with UNC0642 or saline. The procedure of administration is the same as the normal mice. The control mice were given saline in the same procedure.