Ly294002

HUVECs (3 × 10⁵ cells/well for 6-well plates and 6 × 10⁴ cells/well for 24-well plates) were plated overnight in complete endothelial medium containing antibiotics. The next day, the medium was replaced with fresh antibiotics free medium and cells were treated with different concentrations of IL-6, IL-6R, TNF-α and LPS (all from R&D systems, USA) and/or pharmacological inhibitors CP690550, Stattic and LY294002 (all from R&D systems, USA), and PD98059 (Santa Cruz biotechnology, USA) for different time points ranging from 5 min to 48 h. At the end of incubations, supernatants and cells were collected and kept at -80 °C until further analysis. The cells were used to extract total protein or RNA.

HeLa cells were divided into 6 groups: control group (no transfection), NC group (cells transfected with the negative control siRNA), SULF2 siRNA group (cells transfected with SULF2 siRNA), SULF2 group (cells transfected with the SULF2 plasmid), SULF2 + LY294002 group (cells transfected with the SULF2 plasmid followed by treatment with 20 μM LY294002 for 24 h), and SULF2 + U0126 group (cells transfected with the SULF2 plasmid followed by treatment with 25 μM U0126 for 24 h). LY294002 and U0126 were provided by R&D Systems (USA), while SULF2 plasmid, SULF2 siRNA, and NC siRNA were designed and synthesized by Shanghai GeneChem Biotech Co., Ltd. (China) Transfection was performed following the instructions of the Lipofectamine^TM 2000 manufacturer (Sigma, USA).

All drugs were purchased from Sigma (MO, USA) unless otherwise indicated. Stock solutions of 4-aminopyridine (4-AP), pertussis toxin (PTX), cholera toxin (CTX), PMA (Tocris Bioscience, Ellisville, MO) and ω-conotoxin MVIIC (Tocris Bioscience, Ellisville, MO) were prepared in distilled deionized water. Z941 was a kind gift from Dr. Terrance P. Snutch (University of British Columbia, Vancouver, British Columbia, Canada). Stock solutions of cholecystokinin-8 (Tocris Bioscience, Ellisville, MO), LY294002, CCK-4, nifedipine, forskolin, gallein, wortmannin, KT5720 (RD system), devazepide, LY225910, GW5823, BC264, SP600125, SB203580, anisomycin, PP2, PP3, Akt inhibitor III, U0126, and GF109203X were prepared in dimethylsulfoxide (DMSO). The final concentration of DMSO in the bath solution was estimated to be less than 0.01%, and this compound had no functional effects on I_A (not shown).

For hypoxic treatment, BMSCs were cultured in an environment of 5% CO₂, 1% O₂, and 94% N₂ for 10 days, with normoxia as controls. BMSCs that stably overexpressed LIF were conducted through transducing retroviral LIF expression vectors (Promega, USA). To construct BMSCs that stably depleted LIF, lentiviral siRNA vectors (Promega) targeting LIF were transduced into BMSCs. The infected BMSCs were chosen utilizing 2 mg/L puromycin for 14 days. PI3K/Akt signaling was activated by 30 μM agonist 740Y-P (R&D Systems, UK) and blocked by 50 μM inhibitor LY294002 (R&D Systems).

Recombinant human LCN2 (rhLCN2), U0126 and LY294002 were purchased from R&D systems (Minneapolis, MN, USA). LCN2, AKT, phospho-AKT and GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Phospho-ERK1/2 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). ERK1/2 antibody was purchased from Merck Millipore (Merck Millipore, MA, USA). GPX3 and MMD antibodies were purchased from Abcam (Abcam, Cambridge, UK). HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology.

The antibodies that were used for Western blotting analysis and immunostaining were as follows: FOXO1 (#2880 or #2880S, Cell Signaling, Danvers, MA, USA, 1:500); FOXO3 (#9467, Cell Signaling); FOXO4 (#9472, Cell Signaling, 1:500); p-FOXO1 (#ANTY011115, Antagene, Sunnyvale, CA, USA, 1:500); pro-SP-C (#AB3786, Millipore, Billerica, MA, USA, 1:500); AKT (#9272, Cell Signaling, 1:1000); p-AKT (ser473) (#3787, Cell Signaling, 1:1000); Lamin A/C (#SC20681, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:1000); NKX2.1 (#MS-699-P0, Thermo Scientific, Pittsburgh, PA, USA, 1:500); GAPDH (#AM4300, Applied Biosystems, Austin, TX, USA, 1:1000), EIF-4E (#610270, BD Biosciences, San Jose, CA, USA, 1:200); eIF2α (#11386, Santa Cruz Biotechnology, 1:500); β-ACTIN (#ab6276, Abcam, Cambridge, MA, USA, 1:2000); and P180 lamellar body protein (#MMS-645R, Covance, Princeton, NJ, USA, 1:2000). VIIIB2 is a monoclonal antibody (mAb) that is specific for rat AT1 cells in situ that was previously generated in our laboratory (1:500) [76 (link)]. KGF, Ly294002 (PI-3K/AKT inhibitor) and isopropyl β-D-1-thiogalactopyranoside (IPTG) were purchased from R&D (Minneapolis, MN, USA), EMD Biosciences (San Diego, CA, USA) and Sigma, respectively.