Momelotinib

Primary murine WT and PTEN KO CAFs were serum starved overnight before treatment. The next day, inhibitors were added in indicated concentrations (Figs 1 and S1) in serum-free medium and treated for 4 h. Cells were then stimulated with 10% before lysing and protein was collected for downstream Western blotting. The following inhibitors were used: Momelotinib (JAK inhibitor) (Cat. no. S2219; Selleckchem), Pictilisib (pan PI3K inhibitor) (#S1065; Selleckchem), Alpelisib (PI3K-α) (#S2814; Selleckchem), and IPI-3406 (PI3K-γ) (#S7335; Selleckchem). Each experiment was repeated three times.

Cells were plated at a density of 2000–4000 cells/well on laminin-coated 96-well plates in a minimum of triplicates. After three days of incubation, compound was added to each well in concentrations from 0.07–20 µM and incubated at 37 °C, in 5% CO₂, 95% humidity for eight days (192 h). For Activin A / BMP4 studies, cells were supplemented with media containing BMP4 (10 ng/ml, Peprotech, London UK) or Activin A (10 ng/ml, Thermo Fisher) 24 h prior to adding the drug. Drug was added in media supplemented with Activin A and BMP4 in concentrations from 0.07 to 20 µM and incubated at 37 °C, 5% CO₂, 95% humidity for eight days (192 h). Dorsomorphin, DMH1, perhexiline maleate, LDN-193189, LDN-212854 and LDN-214117 were purchased from Sigma-Aldrich; saracatinib and momelotinib were purchased from Selleckchem (Houston, TX, USA); K02288 was purchased from BioFocus (Saffron Walden, UK); LDN-213844 (K03841b), LDN-212838 (K03449c) and K05907 were synthesised at the Structural Genomics Consortium, Oxford (see Supplementary Methods for chemical structures and synthesis). LDN-213819 was a kind gift from Paul Yu and Greg Cuny (Harvard University). Cell viability was assessed by the CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI, USA) and GI₅₀ values were calculated using GraphPad Prism version 6 as the concentration of compound required to reduce cell viability by 50%.

D-luciferin was purchased from Gold Biotechnologies (St. Louis, MO, USA). TNF-α protein was purchased from Abcam (Cambridge, UK) and R-7050 was purchased from Selleck (Houston, TX, USA). Screened compounds are described below. Repurchased hits, including NVP-BSK805, momelotinib, and fedratinib, were obtained from Selleck. ZL181 used in this study was synthesized as previously described [29 (link)]. All salts for electrophysiological recordings were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted.

Pre-steatotic spheroids were subject to drug treatment. The preventive effect of steatosis was assessed with Momelotinib (1, 5 and 10 μM). Momelotinib was purchased from Selleck Chemicals.

Type-I JAK inhibitors rux (CAT#S1378), BMS-911543 (CAT#S7144), AZD-1480 (CAT#S2162), fedratinib (CAT#S2736), momelotinib (CAT#S2219), and pacritinib (CAT#S8057) were all purchased from Selleckchem. Type-II JAK inhibitor, CHZ-868 (CAT#HY-18960) was purchased from MedChemExpress. Inhibitor stocks (10 mM) were diluted in DMSO so that the final concentration of DMSO in culture media and assays was 0.05–0.1%.

PMA (Sigma-Aldrich) was used at 0.4-100 ng/ml and ionomycin (Sigma-Aldrich) was used at a concentration of 100 ng/ml. 3-azido-3-deoxythymidine (zidovudine; AZT) (Sigma-Aldrich) was used at 10 µM, raltegravir at 5 µM and efavirenz at 0.32 µM were obtained from the NIH AIDS Research and Reference Reagent Program. TNFα (Merck) was used at a concentration of 0.08-2 ng/ml and LPS at 100 ng/ml (Merck). Fedratinib, TG101209, AZD1480, AZ960, gandolitinib, WP1066, XL109, ruxolitinib, momelotinib, JQ1 were used at a concentration of 0.2-5 µM, pacritinib at 40 nM-1 µM and Q-VD-Oph at 10 µM (all from Selleckchem). Vorinostat (SAHA) was used at a concentration of 0.2-5 µM (Prochifar srl, Italy) and panobinostat at 3.2-400 nM (LC Laboratories).

Primary human hepatocytes obtained from Massachusetts General Hospital (Boston, MA, USA), In Vitro ADMET Laboratories (Columbia, MD, USA), or Lonza (Switzerland) were plated in collagen-coated wells as per the manufacturers’ instructions and incubated at 37 °C and 5% CO₂ for 4 h to allow attachment. For drug treatments, momelotinib (SelleckChem, cat# S2219) was diluted in DMSO and added to hepatocytes in complete medium for 18 h at 37 °C and 5% CO₂ prior to takedown. The same volume of DMSO was used in control wells. In experiments where BMP2 was used, BMP2 (R&D Systems, Inc., Minneapolis, MN, USA, cat# 355-BM-100) was reconstituted in 0.1% BSA/4 mM HCl to 100 μg/mL. It was used at a final concentration of 6.2 μg/mL for 18 h. For siRNA knockdown, primary hepatocytes were reverse transfected using RNAiMax (Invitrogen, cat# 13778-075) with 10 nM siRNA from Dharmacon (siGENOME). Following an overnight transfection in hepatocyte plating medium (Lonza, cat# MP100), the medium was replaced with complete medium and cultured for an additional 48 h, for a total of 72 h of knockdown.