J4455

Interferon-gamma (IFN-γ, 300-02) and tumor necrosis factor alpha (TNF-α, 300-01A) were purchased from Peprotech. AMPK agonists Metformin hydrochloride (PHR1084), AICAR (A9978), Resveratrol (R5010); SIRT1 agonist SRT1720 (SRT1720) and NRF-2/HO-1 agonist Protoporphyrin IX cobalt chloride (C1900) were purchased from Sigma-Aldrich. Inhibitors of NF-κB Emodin (E7881) and JSH23 (J4455); IKKβ inhibitor IKK16 (SML1138); STAT1 inhibitor Fludarabine (F2773) and NOS2 inhibitor 1400W (W4262) were purchased from Sigma-Aldrich. Inhibitors of MEK1 and MEK2 PD98059 (ab120234), JNK SP600125 (ab120065) and MAPK SB202190 (ab120638) were purchased from Abcam. Reagents were dissolved in DMSO or water (Metformin and AICAR) at 100x stock concentrations and maximum DMSO concentration used in cells was 1%. Monoclonal antibody against 3-nitrotyrosine (ab61392) was purchased from Abcam. Monoclonal antibody against NF-κB p65 (sc-8008) was purchased from Santa Cruz Biotechnology.

After induction of IR injury, mice received an intravitreal injection of recombinant human HMGB1 (1 μg/2 μl; 1690-HM-025, R&D system, Inc., USA), glycyrrhizic acid (120 μM/2 μl; 50531, Sigma Aldrich, USA), nuclear factor κB (NF-κB) inhibitor JSH-23 (20 μM/2 μl; J4455, Sigma Aldrich, USA) , caspase-8 inhibitor Z-IETD-fmk (20 μM/2 μl; Calbiochem, San Diego, CA, USA), and PBS (2 μl) vehicle as sham. Mice were sacrificed and eyes were enucleated 48 h after intravitreal injection.