Kn 92

The detailed experimental design was illustrated in Fig 1. Chemicals other indicated were obtained from Beyotime and Boster (Shanghai, China). Four times injections of fentanyl (60 μg/kg per injection, s.c.) at 15 min intervals, resulting in a cumulative dose of 240 μg/kg [10 (link), 11 (link)] were conducted to induce OIH in this study. The control animals received an equal volume (1.2 ml/kg) of physiological saline. KN93(N-[2-[[[3-(4’-Chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4′-methoxybenzenesulfonamide phosphate salt) and KN92 (2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, monohydrochloride)were purchased from Cayman (Ann Arbor, MI) and were dissolved in 50% dimethyl sulfoxide (DMSO). 50% DMSO was used as a vehicle control. Intrathecal (i.t.) injections were performed manually between the L5 and L6 inter-vertebral space in conscious rats as previously described [12 (link)]. The injection was performed using a glass microsyringe (RWD Life Science, Shenzhen, China). Each rat was injected with a volume of 0.45 μl. Success of the i.t. injection was verified by a lateral tail-flick.

Crocin (Figure 1(a)), LPS (phenol extracted from Salmonella enteritidis), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), actinomycin D (Act. D), cycloheximide (CHX), 2-(3,6-bis(acetyloxy)-2,7-dichloro-9H-xanthen-9-yl)benzoic acid acetoxymethyl ester (Fluo-3/AM), and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cobalt protoporphyrin (CoPP) and zinc protoporphyrin IX (ZnPP) were bought from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). LY294002, PD98059, SP600125, and SB203580 were obtained from A.G. Scientific (San Diego, CA, USA). Nifedipine, 1,2-bis (o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA/AM), and calmidazolium chloride were acquired from Calbiochem (EMD Millipore, Billerica, MA, USA). KN92, KN93, and KN62 were purchased from Cayman Chemical (Ann Arbor, MI, USA) and Calbiochem.

A549 and MSTO-211H cells were provided by ATCC and cultured in DMEM and RPMI (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (R&D Systems, Minneapolis, MN, USA) and 1% L-glutamine (Thermo Fisher Scientific), respectively. Cells were treated with the AKT inhibitor AKTi VIII (Calbiochem, San Diego, CA, USA), epidermal growth factor (EGF) (Invitrogen, Carlsbad, CA, USA), insulin growth factor (IGF)2 (Peprotech, Rocky Hill, NJ, USA), the lysosome inhibitor bafilomycin A1 (Sigma-Aldrich, St. Louis, MO, USA), the CDK4/CDK6 inhibitor Abemaciclib (Selleckchem, Huston, TX, USA), and the following compounds from Cayman Chemicals (Ann Arbor, MI, USA): the MEK1/2 inhibitor U0126, the proteasomal inhibitor MG-132, the CaMK inhibitor KN-93, the KN-93 inactive derivative KN-92, the CaMKK inhibitor ST-609 and the calpain inhibitor 1/ALLN.