4μ8C

4μ8C (IRE1 Inhibitor III) is a highly selective and potent inhibitor of IRE1α ribonuclease (RNase) activity (IC₅₀ ≈ 6–10 μM). By forming a covalent bond with the RNase active site of IRE1α, 4μ8C specifically inhibits its ribonuclease activity, thereby blocking the splicing of XBP1 mRNA. 4μ8C can be used in research on endoplasmic reticulum stress, the unfolded protein response, immune regulation, and tumor and metabolic diseases.

IRE1 Rnase:76 nM

Methods: Human hepatocellular carcinoma cell lines HepG2, Huh7, and SNU449 were pretreated with 10 μM 4μ8C for 2 hours, then transferred to fresh medium containing 1 μM doxorubicin for continued culture for 24 hours. Cell viability was assessed using the MTT assay.
Results: 4μ8C pretreatment significantly enhanced doxorubicin cytotoxicity, with combined treatment groups exhibiting substantially greater reductions in cell viability compared to monotherapy groups. [1]

Methods: Male C57BL/6J mice were intraperitoneally injected with diethylnitrosamine every two weeks for 28 weeks to induce hepatocellular carcinoma. From week 25 to week 28, mice received intraperitoneal injections of 4μ8C (10 mg/kg) twice weekly for 3 weeks, concurrently with intravenous doxorubicin (4 mg/kg) twice weekly.
Results: Combined therapy significantly reduced tumor burden in mice, markedly decreased intracellular triglyceride levels in tumor tissues, and diminished tumor-associated inflammatory responses. [1]
Methods: C57BL/6J mice were fed a high-fat, high-fructose, high-cholesterol diet for 24 weeks to induce NASH. From weeks 20 to 24 of dietary induction, mice received daily intraperitoneal injections of 4μ8C (3.3 mg/kg/day) for 4 consecutive weeks.
Results: Plasma total EV counts decreased significantly, hepatic injury was mitigated, and proinflammatory cytokine expression (TNF-α, IL-1β) was downregulated. [2]

In Vitro IRE1 RNase and RIDD Assays: Analysis of radiolabeled Xbp1 substrate cleavage is performed as previously except that mammalian IRE1 reaction buffer is used. In vitro RIDD substrates are synthesized by in vitro transcription using the T7-MAXIscript Kit in the presence of 32P ATP or Cy5-UTP on templates isolated by RT-PCR from mouse Min6 cells (Ins2) or PCR from cloned XBP1 cDNA. The resulting products are gel purified to obtain full-length substrate. Reactions are then separated by 15% UREA-PAGE for analysis by phosphorimaging or by near-infrared imaging using the LI-COR Odyssey scanner.

Cells are seeded in phenol red-free cell culture medium in 96 or 24 well dishes at a density of 5 × 103 or 5 × 104 cells per well, respectively. Cultures are incubated for 16 h before treatment with 4μ8C for 24 h. Cultures are then analyzed by the addition of 200 μM WST1 and 10 μM phenazine metho-sulfate. After development of the reagent for 2 h at 37°C, the hydrolyzed dye is detected by absorbance at 450 nm, after subtracting background and absorbance at 595 nm. Alternatively, cell viability is determined by staining of the adherent culture with crystal violet. Quantitation of the dye uptake is analyzed by extensive washing of the stained cells with water and solublization of the crystal violet in methanol followed by absorbance measurements at 595 nm. (Only for Reference)

DMSO: 33.33 mg/mL (163.24 mM), Sonication is recommended.