Ly294002

ALP staining was performed to evaluate osteoblastogenic activity (29 (link)). hFOB1.19 osteoblasts (5×10⁴ cells/well) were cultured in a 6-well plate and treated with TGF-β1 (0, 0.5, 1, 2, 5 and 10 ng/ml), in the presence or absence of a PI3K/AKT inhibitor (10 µM LY294002; PeproTech, Inc., Rocky Hill, NJ, USA) or an inhibitor of mammalian target of rapamycin/S6 kinase 1 (mTOR/S6K1; 100 nM rapamycin) (30 (link)). Following incubation for 7 days at 37°C, osteoblasts were washed with PBS two times, fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature and washed with deionised water twice. Cells were incubated with a 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) ALP colour development kit (Beyotime Institute of Biotechnology, Haimen, China) for 1 h at 37°C and washed with deionised water two times. Then, images were acquired using a phase contrast microscope equipped with a charge-coupled device (CCD) camera (magnification, ×100). Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) was used to evaluate the percentage of the total stained area.

SFN and H₂O₂ were purchased from Sigma-Aldrich (St. Louis, MO, USA). All antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Cell culture reagents were offered from Gibco (Grand Island, NY, USA). Inhibitor of phosphatidylinositol 3-kinase (PI3k), LY294002, was purchased from PeproTech (NJ, USA).

Primary human OFs were isolated and cultured using established explant techniques.7 (link) Samples were from either TED patients undergoing orbital decompression surgery (herein referred to as Graves' OF or GOFs) or non-TED patients undergoing unrelated orbital surgery (herein referred to as non-TED or NOFs) at the Flaum Eye Institute. The non-TED patients did not have any inflammatory orbital diseases. Relevant clinical information including history of smoking, steroid use, and radiation treatment are listed in the Table. Once established, fibroblasts strains were characterized as previously described.3 (link),8 (link),31 (link) Sample collection followed the tenets of the Declaration of Helsinki and were approved by the Research Subjects Review Board at the University of Rochester Medical Center. Informed, written consent was obtained from all patients before surgeries. Explanted OFs were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. All media and supplements were from Gibco (Carlsbad, CA, USA) and FBS was from Hyclone (Logan, UT, USA). LY294002, a specific PI3K/Akt inhibitor, was from Peprotech (Rocky Hill, NJ, USA).