Ly294002

BD Matrigel™ Basement Membrane Matrix, culture dishes, and plates were obtained from Corning Inc. (Corning, NY, USA). Concanavalin A (Con A), sodium dodecyl sulphate (SDS), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Endothelial Cell Medium (ECM) was obtained from ScienCell (Carlsbad, CA, USA). PD98059 and LY294002 were the products of Tocris Bioscience (Bristol, UK). The primary antibodies, such as rabbit anti-phospho-Akt (Ser473) (#4060S), rabbit anti-Akt (#9272S), rabbit anti-phospho-ERK1/2 (#4730S), rabbit anti-ERK1/2 (#4695S), rabbit anti-phospho-p38 (#4511S), rabbit anti-p38 (#8690S), mouse anti-p27 (#3686S), mouse anti-p21 (#2947S), rabbit anti-cyclin D1 (#55506S), and rabbit anti-cyclin E (#20808S) antibodies were from Cell Signalling Technology (Beverly, MA, USA). The polyvinylidene fluoride (PVDF) membranes were the products of Millipore (Billerica, MA, USA). EdU Apollo®488 In Vitro Imaging Kit was a product of RIBOBIO (Guangzhou, China).

Anti-GAPDH antibody was purchased from Fitzgerald Antibodies (Acton, MA) and was used at a dilution of 1:1,000. Anti-phospho-Akt (Ser473), anti-Akt, and anti-phospho-eNOS (Ser1177) antibodies were purchased from Cell Signaling (Danvers, MA) and were all used at a dilution of 1:1,000. Anti-eNOS antibody (1:500 dilution), anti-mouse IgG (1:10,000 dilution), and anti-rabbit IgG (1:2,000 dilution) antibodies were purchased from Abcam (Cambridge, MA). MK2206 and GDC0068 were purchased from SelleckChem (Houston, TX). Gallein, LY294002, L-NAME, and ODQ were purchased from Tocris (Ellisville, MO). All other chemicals were purchased from Sigma (St. Louis, MO).

For chemical treatments, 20 5-dpf zebrafish larvae fed with 10% cholesterol-infused diet were co-exposed with either 1 μM of JSI124, a Stat3 inhibitor (Tocris), or LY294002, a PI3K inhibitor (Tocris), for 7 days. For induction of tgfb1a expression from tgfb1a+ transgenic zebrafish, 1, 2 or 3 μM of mifeprisone (Sigma) were used to incubate 3-month-old fish for 6 weeks. For inhibition of Smad and Erk activity, 1 μM PD169316 (Sigma) or U0126 (Sigma) was added to tgfb1a+ zebrafish after 3 weeks of 3 μM mifepristone induction. All chemical dosages were selected based on the highest all-survival concentrations.

Dulbecco’s modified Eagles’ medium (DMEM), penicillin/streptomycin, trypsin/EDTA, fetal bovine serum (FBS), KGM-SFM medium, and phosphate-buffered saline (PBS) were from Gibco (Life Technologies, USA). Arecoline, melatonin and MTT were from Sigma (Sigma-Aldrich Chemical Company, St. Louis, MO, USA). ELISA kits for MMP-9 were from PeproTech (PeproTech, Inc., Rocky Hill, NJ, USA). Catalase, SB431542, 5Z-7-Oxozeaenol, PD153035, AG490, U0126, LY294002, and aspirin were purchased from Tocris. Phospho-TAK1 (p-TAK1) antibody was from Abcam (ab79583). Antibodies for TGF-β1, and GAPDH were from Santa Cruz. P-Smad2 antibody was from Cell Signaling Technology. PBL extract and ANE were prepared as before [8 (link), 12 (link), 13 (link), 21 (link)]. HC was synthesized as previously [22 (link)].

Reagents were purchased and used as follows: GSK269962A (1 μM; Axon Medchem) in dimethyl sulfoxide (DMSO; #1167, Axon) was used as a ROCKi. LY-294002 (10 μM; #1130, Tocris Bioscience) in DMSO was used as a PI3K inhibitor. Adenosine (5 μM; A4036, Sigma-Aldrich) in DMSO, APCP (10 to 100 μM; M3763, Sigma-Aldrich), and anti-CD73 antibody (clone 2C5, Medimmune/Astra-Zeneca) were used. Time of treatment and drug concentration were specified in each experiment.

MSCs and ACs were cultured as 3D pellets (5 × 10⁵ cells/pellet) in chondrogenic medium consisting of high-glucose DMEM, 0.1 µM dexamethasone, 0.17 mM ascorbic acid-2 phosphate, 4 mM sodium pyruvate, 0.35 mM proline, 5 µg/mL transferrin, 5 ng/mL selenous acid, 1.25 mg/mL bovine serum albumin (all from Sigma-Aldrich, St. Louis, MO, USA), 1% penicillin/streptomycin, 1% ITS+ premix (Corning Life Sciences, New York City, NY, USA) or similar amounts of insulin (Lantus, Sanofi-Aventis, Frankfurt, Germany), and 10 ng/mL recombinant human TGFβ1 (Biomol, Hamburg, Germany) for up to 6 weeks with medium changes three times a week.
For the indicated time points during MSC chondrogenesis, the chondrogenic medium was supplemented with the BMP inhibitor LDN-212854 (LDN-21; 500 nM in DMSO solvent, day 0–42; Sigma-Aldrich, St. Louis, MO, USA), the WNT-inhibitor IWP-2 (2 μM in DMSO solvent, day 14–42), or LY294002 (LY; 0.25 µM–25 µM in DMSO, day 21–42; both from Tocris Bioscience, Bristol, United Kingdom). When appropriate, the controls were treated with DMSO. Where indicated, TGFβ1 was withdrawn from day 21 onward. For the Western blot detection of phosphorylated AKT, pellets were harvested at the designated days 48 h after the last medium exchange.

Tamoxifen was purchased from Nacalai Tesque (Tokyo, Japan). Caffeine, caffeic acid, chlorogenic acid, pyrocatechol, and trigonelline were purchased from Sigma-Aldrich (St. Louis, MO). LY294002 and U0126 were purchased from Tocris Bioscience (Bristol, UK). Anti-ERα, anti-phospho-MEK1/2 (S217/221), anti-MEK1/2, anti-phospho-ERK1/2 (T202/Y204), anti-ERK1/2, anti-phospho-Akt (S473), anti-Akt, and anti-cleaved caspase-3 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-p53, anti-cyclin D1, and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Peroxidase-conjugated rabbit anti-mouse, goat anti-rabbit, and swine anti-goat secondary antibodies were from Dako (Glostrup, Denmark).