Ldn193189

RPMI 1640 and pancreatin were from Gibco (USA). Fetal bovine serum (FBS) was from TransGen (China). Trizol^TM reagent was from Life (USA). PrimeScript^TM RT reagent kit with gDNA eraser was from TaKaRa (Japan). FastStart universal SYBR green master and BrdU assay ELISA kit were from Roche (Switzerland). Annexin V-FITC/propidium iodide apoptosis detection kit, Hoechst 33258 and cell cycle detection kit were from KeyGEN (China). Agilent M × 3005P real-time PCR machine was from Agilent (USA). LDN-193189 was from Selleckchem (USA). FACSCalibur flow cytometry was from BD Biosciences (USA). Olympus IX71-A12FL/PH fluorescence microscope and JEOL JEM-1200EX transmission electron microscopy were from Japan. Microplate reader and Nanodrop 2000 were from Thermo Scientific (USA).

MB109 is a recombinantly expressed human BMP-9 cytokine. It contains a methionine residue in front of the mature domain of human BMP-9 (Ser320-Arg429, 110 residues). MB109 was prepared as described previously [13 ]. Recombinant BMP-2 was purchased from joint Protein Central (jointproteincentral.com, Incheon, Korea). The antibodies against p21, P-SMAD1/5/8 and P-p38 were purchased from Cell Signaling Technology (Massachusetts, USA), ID3 from Santa Cruz Biotechnology (California, USA), β-actin from Sigma Aldrich (Missouri, USA), CD90 from Novus Biologicals (CO, USA), AFP from Abcam (Cambridge, UK), PE-conjugated CD90 from BD Biosciences (New Jersey, USA) and FITC-conjugated CD44 from Miltenyi Biotec (Bergisch Gladbach, Germany). Chemical inhibitors, LDN193189 was purchased from Sellekchem (Texas, USA) and SB202190 was purchased from Sigma Aldrich (Missouri, USA). The cell culture media, fetal bovine serum (FBS), Penicillin-Streptomycin Solution (P/S) and Trypsin-EDTA were purchased from Hyclone (Utah, USA).

Generally, MEFs were treated with LDN193189 (selleck S2618, 0.25 μM), SB431542 (selleck S1067, 1 μM) on day 1 and day 2. CHIR99021 (selleck S2924, 3 μM), DAPT (selleck S2215, 5 μM), and VPA (sigma P4543, 0.5 mM) were used on day 3 and day 4. CHIR99021 (selleck S2924,3 μM), DAPT (selleck S2215, 5 μM) were used on day 5 and day 6. Shh (Peprotech 315225, 100 ng/ml) and Purmorphamine (selleck S3042, 1 μM) were used to improved neural differentiation on day 7 and day 8. Then, all small molecules were withdrawn on the last 2 days. After 10 days, SMSINS cells were plate into coated poly-D-lysine (10 μg/ml)/laminin (5 μg/ml) (sigma) (PDL/L) for proliferation or next test. However, for neurosphere culture, SMSINS cells were trypsinized to single cells and seeded into uncoated 24 well-plates in NSC medium. The half of medium was switched every 2 days.
To mediate mTOR signaling, we repeated the program except that Rapamycin (sigma V900930, 2 μM) was added on day 3 and day 4 and MHY1485 (selleck S7811, 1 μM) replaced VPA.

BMSCs were induced and cultured in osteogenic medium according to a StemPro Osteogenesis Differentiation Kit (Invitrogen, USA). Recombinant human BMP-4 protein (rhBMP-4) (R&D Systems, USA), a Smad inhibitor (LDN-193189) (Selleck, USA), and metformin (TargetMol, USA) were separately added to 0.5 ml medium in 24-well dishes (Corning, USA), and the final concentrations of each substance in the medium were as follows: rhBMP-4 (+: 10 ng/ml; ++: 50 ng/ml), LDN-193189 (+: 0.1 μM; ++: 0.5 μM), and metformin (+: 30 μM; ++: 100 μM). After 10 d of induction, cells were fixed in 70% ethanol for 1 h and stained using an ALP staining kit (Beyotime, China) according to the manufacturer's protocol. Intracellular ALP activity assays of BMSCs were performed at 3, 5, and 7 d of induction using an ALP Activity Assay Kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's protocol and were standardized based on protein concentration. After induction for 21 d, cells were fixed in 70% ethanol and stained with 2% alizarin red S staining solution (Sigma-Aldrich, USA) for 5 min. Then, 1 ml of isopropanol was added into each well to dissolve the red perylenequinone derivatives in the calcium nodules, and the optical density (OD) values were measured at a wavelength of 550 nm.

GCDCA (Purity 97%) was purchased from Shanghai Yuanye Bio-Technology Company (Shanghai, China). It was dissolved in cell medium and stored at 4 °C before used. LDN-193189 and GW4064 were purchased from Selleck Chemicals Company (Houston, TX, USA). Guggulsterone was purchased from MedChemExpress Company (Shanghai, China). LDN-193189, GW4064 and guggulsterone were dissolved in DMSO and stored at −20 °C before used.

To influence blood flow, control or ablated Tg(vmhc:mCherry-NTR) larvae were incubated in E3 water with 1.8 mM Tricaine (3-aminobenzoic acid ethyl ester, Sigma, St. Louis, MO, USA) or 10 mM BDM (2,3-butanedione monoxime, Sigma, St. Louis, MO, USA) directly after ablation for 15 h [43 (link)]. For signaling pathway studies, zebrafish larvae were incubated in E3 water with different drugs or solvent right after ablation for 20 h. A quantity of 100 μM of DAPT (Sigma, St. Louis, MO, USA) was used to inhibit Notch signaling [5 (link)]; 5 μM Cardiomogen-1 (Sigma, St. Louis, MO, USA) was used to inhibit Wnt signaling [51 (link)]; 7.5 μM K02288 (Selleck, Houston, TX, USA) or 5 μM LDN193189 (Selleck, Houston, TX, USA) was used to inhibit Bmp signaling [53 (link),55 (link)]; 1 μM RA (Sigma, St. Louis, MO, USA) was used to activate RA signaling [69 (link)]; 5 μM PD153035 (Selleck, Houston, TX, USA) was used to inhibit EGF signaling [70 (link)].