Dorsomorphin

Confluent H9 hESCs were split with 0.02% EDTA/PBS (Sigma) in 1 : 5 ratios into culture dishes coated with Geltrex (Invitrogen) and cultured in N2B27 medium (all supplements from Gibco), supplemented with 5 μM Dorsomorphin (Sigma). At this stage, cells were defined as passage 1 (P1) and the cell was called hES-NP cells. In the neural differentiation, the cells were consistently cultured in the Geltrex-coated culture plates. Cells of P1 and P2 were split by 0.02% EDTA/PBS (Sigma) into small clumps, similar to hESC culture, and continuously cultured in N2B27 medium plus Dorsomorphin. From P3, cells were dissociated into single cells by TrypLE express (Gibco), and Dorsomorphin could be withdrawn from N2B27 medium with the addition of 10 ng/mL bFGF. The cells were able to survive in long-term culture. hES-NP cells gradually change their morphology and gene expression profile, when cultured over passage 30, and we termed these cells as hES-neural stem cells. To induce postmitotic cell types, hES-NP cells were cultured in the basal medium without growth factors for 2 weeks.

After 24 h, to allow cell attachment, the medium was replaced and both the cell lines were incubated for 1 h with Sirtinol (Sirt, a sirtuins inhibitor; Tocris Bio-Techne, Minneapolis, MN, USA) or Compound C (CC, also known as dorsomorphin, an AMPK inhibitor; Merck KGaA, Darmstadt, Germany) at the concentration of 10 μM, or both, or their vehicle. After 1 h of incubation, Taurisolo^® 100 μg/mL or vehicle (culture medium) was added and incubated for 1 h. After 1 h, the cells were incubated for 2 h with the pro-oxidant agent represented by H₂O₂ (200 μM for HASMCs and 100 μM for HUVECs). Cell viability was assessed using an aqueous solution of the cell proliferation reagent WST-1 (Roche, Basilea, Switzerland), (see Supplemental Materials).

Cortical organoids were generated from hPSC lines using a reported protocol^{69 (link)}, with some modifications. Briefly, hPSCs were dissociated to single cells by exposed to Accutase for 5 mins at 37 ℃. Collect cells and plate 8000 cells in each well of PrimeSurface 96 V Plate (Sumitomo Bakelite, MS-9096VZ) to form equal prime EBs, followed by careful centrifuge at 300 × g for 3 min. Wells contained neuronal induction medium containing 50% NIM (DMEM/F12, 1× N2 supplement, 1× NEAA, 1× GlutaMAX, Heparin (Sigma, 2ug/ml)) and 50% Essential 8 medium with dorsomorphin (compound C; Merck, 5 µM), SB-431542 (10 μM) and Y-27632 (10 μM) for first six days. The medium was changed on day 7 with neural medium, containing Neurobasal, 1× B-27 supplement and 1× GlutaMAX, which were supplemented with 10 ng/ml FGF-basic and 20 ng/ml EGF. To promote growth and differentiation, organoids were transferred into ultra-low-attachment 10 cm dishes on day 8 using a cut 1000 μl pipette tip (Nunc). These organoids were cultured using a Sunflower Mini-Shaker (Grant-Bio) with every other day medium change (day 8 to day 22). To promote neural progenitors differentiation, organoids subsequently cultured in neural medium was supplemented with 10 ng/ml BDNF, 10 ng/ml GDNF, 10 ng/ml IGF-1 and 20 ng/ml NT3 starting at day 23. Medium changes every 3 or 4 days.

iPSCs were dissociated using 0.5 mM EDTA and transferred to ultra-low attachment culture dishes in E8 medium (without TGF-β1 and FGF2) for embryoid body (EB) formation. After 2 days, EBs were transferred to Matrigel-coated dishes. The medium was changed to a differentiation medium: DMEM/F12 containing 20% FBS, L-glutamine, and non-essential amino acids for mesoderm and endoderm induction; or DMEM/F12 containing 50% neurobasal medium, B27, N2, and L-glutamine for ectoderm induction. After 14 days, the cells were fixed and analyzed by immunofluorescence staining with antibodies against TUJ1, SMA, and AFP. For the directed differentiation of endoderm, cells were treated with RPMI supplemented with 2% FBS, 100 ng/mL activin A (R&D systems, Minneapolis, MN, USA) and 5 µM CHIR 99021 (LC laboratories, Woburn, MA, USA) for the first day, followed by the same media without CHIR99021 for 2 days. For mesoderm, cells were treated with advanced RPMI (Thermo Scientific) supplemented with Glutamax (Thermo Scientific) and 5 µM CHIR 99021 for 2 days. For ectoderm, cells were treated with DMEM (Thermo Scientific) supplemented with 1 µM dorsomorphin (Merck), 10 µM SB431542 (Merck), and 1 µM PD0325901 (Tocris, Bristol, UK) for 8 days. Germ layer-specific primers listed in Supplementary Table S1 were used to determine the gene expressions by ΔΔCt method.

ASCs were seeded in flasks or multiwell plates at a density of 7.5 × 10³ cells/cm² (70%–80% confluence) and allowed to adhere for 24 h. Cells were washed twice in PBS and serum starved in FBS‐free standard culture medium (SFM) for a further 24 h. On the day of stimulation, medium was aspirated, cells washed twice in PBS and stimulated with 100 ng/mL of GDF6 (Peprotech) in SFM as previously described.
³ (link),
⁶ (link),
⁷ (link) Stimulation was performed for a variety of durations which are described for each experiment and forms the temporal‐nature of these studies.
To assess the importance of intracellular signaling mechanisms on the early response genes following GDF6 stimulation, Smad1/5/8 phosphorylation was inhibited by pre‐incubation with 10 μM dorsomorphin (Merck, cat no. 171261) and ERK1/2 phosphorylation was inhibited by pre‐incubation with 10 μM U0126 (Merck, cat no. 662009).
⁷ (link) Furthermore, protein translation was inhibited by pre‐incubation with 10 μg/mL cyclohexamide (Sigma, cat no. C4859) to help elucidate whether gene expression was directly downstream of GDF6‐mediated signaling or reliant upon de novo protein synthesis. In all experiments, following stimulation, cells were washed twice with ice‐cold PBS before either protein or RNA extraction.

Forelimb buds from two mouse embryos (E10.5-E10.75) were incubated in ice-cold 2% trypsin (Gibco)/PBS at 4°C for 30 min and the digestion was stopped by an excess of DMEM medium with 10% fetal bovine serum (FBS). The limb bud ectoderm was manually removed and mesenchymal cells dissociated by gentle pipetting. Cells were plated in four wells of a 96-well plate in high-glucose DMEM medium (10% FBS, 4.5 g/l glucose, 100 U penicillin, 0.1 mg/ml streptomycin and 200 mM L-glutamine, Merck). After 8-9 h, non-adherent cells were removed by changing the medium. Two wells were treated with either 20 μM cyclopamine KAAD (dissolved in ethanol; Calbiochem), 300 ng/μl FGF8b (dissolved in PBS, 0.1%BSA; R&D), 5 μM dorsomorphin (dissolved in DMSO; Merck) or with 20 ng/ml BMP4 (dissolved 4 mM HCl; R&D) for 24 h, while others served as controls (normal medium with solvent). After 12 h, cells were gently detached using trypsin and either processed for FACS analysis of specific cell populations (see before) or processed for RT-qPCR analysis (see below). The Wilcoxon test was used to statistically verify differences observed.

HepG2 cell lines were obtained from CLS services Germany. HepG2 cells were grown at 37 °C in an atmosphere of 5% CO₂, 95% air in cell culture dish using 10 mL of DMEM–F-12 medium with 10% fetal calf serum, 1% penicillin, 1% streptomycin, and 1% fungizone. Cells were plated at a split ratio of 1:4. The preconfluent cells were left either untreated (control cells) or pretreated with oleic acid (BSA conjugated oleic acid, Sigma cat no. O3008-5ML), at a concentration of 250 μM for 12 h. Subsequent treatment with BA (Sigma-Aldrich, cat no. 48305-50G-F) was performed in serum free DMEM–F-12 medium. Similarly, for inhibitor assays, the cells were pretreated with Dorsomorphin (AMPK inhibitor, Merck, P5499, 10 µM) for 1 h prior to treatment with the BA. The plasmid pCH9/200LMS is a replication-defective variant of plasmid pCH9/3091 [31 (link)] and encodes the HBV surface proteins under their natural promoters. HepG2 cells with 80% confluence were transfected with and without the plasmid pCH9/200LMS for 48 h, followed by treatment with oleic acid for 12 h. After that treated with bile acid in serum free DMEM for the next 24 h.