Gsk j4

A549, H1703, and H1975 cells were from ATCC. All cell lines were grown in RPMI medium with 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 µg/mL). Cells were plated 48 h before treatment. Cisplatin was obtained from Sigma Aldrich (St. Louis, MO, USA). ML324, JIB04, GSK-J4 and Paclitaxel were from Selleck Chemicals (Houston, TX, USA). TAME is from Cayman Chemical (Ann Arbor, MI, USA).

We used DZNep (Selleck, S7120) , GSK126 (Selleck, S7061) and GSK-J4 (Selleck, S7070) as inhibitors of EZH2 and JMJD3 respectively, which are with DMSO (Dimethyl Sulfoxide) solvent, and used DMSO as negative control. Rat primary HSCs were treated with DZNep (1 μM), GSK126 (5 or 10 μM) or GSK-J4 (5 μM). DZNep and GSK126 treatment were also conducted in JS1 and LX-2 cell lines. Experimentally diseased mice (as below) were administered with DZNep (1 mg/kg) through intraperitoneal injection twice a week. The H3K27me2/3 levels in HSCs and in liver tissue were analyzed by Western blot to ascertain the inhibitory effect.

Immortalized human mammary epithelial cells (HMLE), including cells expressing the empty vector (pWZL), Snail or Twist were maintained as in Mani et. al [4 (link)]. MCF10A cells were cultured in 2D as a monolayer, as described in Debnath et al. [67 (link)]. 4T1 cells were cultured as in Yang et al. [68 (link)]. MCF7 and MDA-MB-231 cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum and penicillin/streptomycin. TGFB (R&D Biosystems, Minneapolis, MN, USA) was reconstituted in sterile 4 mM HCl containing 0.1% bovine serum albumin. TGFB was added to the culture medium at a concentration of 2.5 ng/ml unless otherwise indicated, with the reconstitution solution serving as a vehicle control. GSK-J4 (Selleckchem, Houston, TX, USA) was added to the culture medium at a concentration of 50 nM, with DMSO as a vehicle control.

The murine macrophage-like RAW 264.7 cell line was obtained from the American Type Culture Collection (Maryland, USA); Antibodies against Kdm6a, Kdm6b, H3, H3k27me3 and GAPDH were purchased from Cell Signalling Technology (Beverly, MA, USA); GSDMD was obtained from Abcam (London, UK); Caspase 1 antibody and goat anti-rabbit IgG (H + L) FITC were generated by ABclonal Biotechnology (Wuhan, China); The Kdm6a and Kdm6b inhibitor GSK-J4 was purchased from Selleck Chemicals LLC (Houston, TX, USA); APC anti-mouse CD11b antibody and PE anti-mouse F4/80 antibody were purchased from BD Biosciences (New Jersey, USA); Foetal bovine serum was purchased from Life Technologies (now Thermo Fisher Scientific Inc, NY, USA); Zol was generated by Novartis Pharma Schweiz AG (Basel, Switzerland).

All zebrafish animal experiments were performed following the institutional guidelines approved by the Institutional Animal Care and Use Committee of Fudan University, Shanghai. GSK-J4 (Selleck) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) at a stock concentration of 10 mM and further diluted to the desired concentrations in fresh egg water. DMSO was selected as the vehicle control group. GSK-J1, inactive control compound GSK-J2, and the MAPK/ERK kinase inhibitor U0126 were purchased from MedChem Express. For hair cell damage, neomycin sulfate (Sigma-Aldrich) was added to 5 days post-fertilization (dpf) larvae at a final concentration of 400 μM and incubated for 1 h. This was followed by three rinses in fresh egg water, and the zebrafish larvae were allowed to recover for 24 h or 48 h at 28.5°C. Drug solutions were replaced after 24 h. The larval zebrafish were then fixed with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4°C until further processing.

THP-1 cell lines were cultured in RPMI supplemented with 10% FCS, 100 μg ml⁻¹ of penicillin and 100 μg ml⁻¹ of streptomycin and 1 μg ml⁻¹ puromycin. ABT-199 was purchased from ChemieTek (Indianopolis, IN). Cantharidin, digoxigenin, proscardillin, wortmannin, SB203580, TOFA, IC 261 and vorinostat were all purchased from Sigma. ABT-737, GSK-J4, GSK-126, G007-LK, MM-102, SKI-606 and JNK-IN-8 were purchased from Sellekchem (Houston, TX). LB100 (HY-18597) was obtained from MedChemExpress (MonMouth Junction, NJ). After 4 days of Dox induction (or control without Dox), cells were plated in RPMI 20% foetal bovine serum at 2 × 10⁵ ml⁻¹ in 96-well plate with twofold dilutions of each drug performed in duplicate. At 72 h, cell viability was measured using a plate-reader after addition of 10% Presto Blue Cell Viability Reagent (ThermoFisher Scientific) at emission fluorescence 590 nm. IC₅₀ curves were calculated for each drug in the presence and absence of Dox using GraphPad Prism 6.0 (dose response inhibition) and the difference in IC₅₀ was plotted as a percentage of control (no dox). For ACACA validation, transduced THP-1 cells were induced with Dox for 3 days, then washed into low serum RPMI (0.5%) and cultured at low cell density for 7 days +/−TOFA before cell growth was measured with Presto Blue on a plate reader.