Ciliobrevin a

The mouse preosteoblast cell line MC3T3-E1 (CRL-2593) (ATCC, Manassas, VA, USA) was grown in α-minimal essential (α-MEM) medium supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin and was maintained at 37 °C with 5% CO₂. SB203580 (Ciliobrevin A, Selleckchem, Houston, TX, USA), an inhibitor of phosphorylated p38, was dissolved in DMSO as a stock solution, and the cells were treated with the inhibitor at a final concentration of 20 μM for 16 h, after which the cells were harvested for Western blot analysis.

Dimethyloxallyl Glycine (DMOG, 71210) was from Cayman Chemical (Ann Arbor, MI, USA). N-acetyl-L-cysteine (NAC), hydrogen peroxide (H₂O₂), MTT [3(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide], sodium butyrate and 4′,6-diamidno-2-phenylinole (DAPI) were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). 2’,7’-dichlorofluorescin diacetate (DCF-DA) was purchased from Molecular Probe (Eugene, OR, USA). Ciliobrevin A was from Selleck Chemicals (Houston, TX, USA). PD98059 was from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Mouse antibodies which are reactive with acetylated tubulin (T7451), and β-tubulin (T4026) were from Sigma-Aldrich Co. (St. Louis, MO, USA). Rabbit antibodies which are reactive with Nrf2 and HIF-1α were from Cell signaling Technology (Cat# 12721, Danvers, MA, USA) and Novus Biologicals (Centennial, CO, USA), respectively. Rabbit antibodies which are reactive with Arl13b (17711-1-AP) were from Proteintech Group Inc. (Rosemont, IL, USA). Chicken anti-mouse IgG-Alexa 488 (A-21200) and goat anti-rabbit-Alexa 568 (A-11011) were obtained from Invitrogen (Calsbad, CA, USA). Except where indicated, all other materials are obtained from the Sigma Chemical Co. (St. Louis, MO, USA).

The cells were allowed to grow for 24h after seeding. Subsequently, they were treated with different concentrations of LDC3/Dynarrestin (diluted in culture medium) for either 1h, 6h, 24h or 48h.
For the activation of NFκB cells were incubated with TNFα (Recombinant Human TNF-α, Peprotech Germany, Hamburg Germany, Cat.# 300-01A) for 6h in a concentration of 100ng/ml. The NFκB inhibition was performed with Ammonium pyrrolidine dithiocarbamate (Sigma-Aldrich, Cat.# P 8765, Munich, Germany) for 6h in a concentration of 50μM. In the case of dual incubation, both compounds were applied at the same time.
The cells were also treated with Sorafenib (LC Laboratories, Woburn, USA, Cat.# S-8599), Dasatinib (LC Laboratories, Woburn, USA, Cat.#. D-3307), 1B Inhibitor (Calbiochem Cat.# 539741) and Ciliobrevin A (Selleckchem, Cat.# S8249).