JNK-IN-8

JNK-IN-8 (JNK Inhibitor XVI) is an effective JNK inhibitor that inhibits JNK1, JNK2, and JNK3 with IC₅₀ values of 4.7, 18.7, and 1 nM, respectively. JNK-IN-8 offers the advantages of minimal off-target activity, sustained inhibition, and high specificity. JNK-IN-8 primarily acts by blocking the JNK/c-Jun signaling pathway, inhibiting the phosphorylation of the downstream transcription factor c-Jun, thereby regulating the expression of genes associated with inflammatory responses, apoptosis, and stress responses. JNK-IN-8 can be used in research on tumors, inflammation, and neurological diseases.

JNK1:4.7 nM, JNK2:18.7 nM, JNK:1 nM

Methods: PDAC cells were seeded in 6-well plates for long-term culture. Every 3 days, the medium was replaced with JNK-IN-8-containing medium (1 μM) for 14 consecutive days, followed by crystal violet staining for colony counting.
Results: JNK-IN-8 monotherapy inhibited colony formation. [1]

Methods: P411-T1 or P422-T1 PDX tumors were transplanted subcutaneously into nude mice. Once tumors became palpable, mice were randomly assigned to receive oral gavage of JNK-IN-8 (30 mg/kg, once daily) for 14 days.
Results: Consistent with in vitro findings, JNK-IN-8 potentiated the in vivo antitumor activity of FOLFOX. [1]
Methods: Adult male Sprague-Dawley rats were subjected to tracheal instillation of LPS (5 mg/kg) to establish an ARDS model. Twenty-four hours later, intraperitoneal injections of JNK-IN-8 (20 mg/kg) were initiated once daily. Efficacy was evaluated through behavioral testing and molecular biological analysis post-administration.
Results: JNK-IN-8 treatment significantly shortened the escape latency in ARDS rats, improved spatial learning and memory abilities, and markedly reduced levels of proinflammatory cytokines such as TNF-α, IL-6, and IL-1β in hippocampal tissue.[2]

A375 cells are pre-treated with 1 μM JNK-IN-8 for the indicated amounts of time. Remove the medium and wash 3 times with PBS. Resuspend the cell pellet with 1 mL Lysis Buffer (1% NP-40, 1% CHAPS, 25 mM Tris, 150 mM NaCl, Phosphatase Inhibitor Cocktail, and Protease Inhibitor Cocktail). Rotate end-to-end for 30 min at 4°C. Lysates are cleared by centrifugation at 14000 rpm for 15 min in the Eppendorf. The cleared lysates gel filtered into Kinase Buffer (0.1% NP-40, 20 mM HEPES, 150 mM NaCl, Phosphatase Inhibitor Cocktail, Protease Inhibitor Cocktail) using Bio-Rad 10DG colums. The total protein concentration of the gel-filtered lysate should be around 5-15 mg/mL. Cell lysate is labeled with the probe from ActivX at 5 μM for 1 hour. Samples are reduced with DTT, and cysteines are blocked with iodoacetamide and gel filtered to remove excess reagents and exchange the buffer. Add 1 volume of 2X Binding Buffer (2% Triton-100, 1% NP-40, 2 mM EDTA, 2X PBS) and 50 μL streptavidin bead slurry and rotate end-to-end for 2 hours, centrifuge at 7000 rpm for 2 min. Wash 3 times with 1X Binding Buffer and 3 times with PBS. Add 30 μL 1X sample buffer to beads, heat samples at 95°C for 10 min. Run samples on an SDS-PAGE gel at 110V. After transferred, the membrane is immunoblotted with JNK antibody[1].

JNK-IN-8 is dissolved in DMSO and stored, and then diluted with appropriate media before use[1]. HEK-293 cells stably expressing Interleukin Receptor 1 (HEK293-IL1R) are cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% FBS, 2 mM glutamine and 1×antimycotic/antibiotic solution. Cells are serum starved for 18 h before incubation with DMSO or JNK-IN-8, stimulated with 2 μM Anisomycin for 1h and lysates are clarified by centrifugation for 10 min at 16000 g and 4°C[1].

DMSO: 56 mg/mL (110.33 mM), Sonication is recommended.

H2O: < 1 mg/mL (insoluble or slightly soluble)

Ethanol: < 1 mg/mL (insoluble or slightly soluble)

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 2 mg/mL (3.94 mM), Sonication is recommended.