Nexrutine® (NX) was obtained from Regorafenib supplier Next Pharmaceuticals (Irvine, CA). Stock solution of NX was prepared by dissolving NX in dimethylsulfoxide (DMSO) at a concentration of 1.0 mg/ml. The stock solution was further diluted either in milli Q water or culture medium to obtain various working concentrations. Antibodies specific for cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were procured from Cayman Chemical Company (Ann Arbor, MI). Antibodies specific for ERK1/2, p38, JNK, CDK2, CDK4, p27, p53, p21, cytochrome c, cyclin E1, cyclin

D1 and β-Actin-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), while p-ERK1/2, p-p38, p-JNK, Bax, Bcl-2, cleaved-caspase 3, cleaved-caspase 9, and proliferating cell nuclear antigen (PCNA) were purchased from cell signaling (Beverly, MA). 2-Acetylaminofluorene (2-AAF), 2-β mercaptoethanol (BME), bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), diethylnitrosamine

(DEN), dithiothreitol (DTT), Dulbecco’s Modified Eagle’s Medium (DMEM), Fetal bovine serum (FBS), streptomycin, penicillin, ethylenediaminetetraacetic acid (EDTA) disodium salt, trypsin/EDTA solution, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phenylmethylsulphonyl fluoride (PMSF), propidium iodide (PI), RNase A, protease inhibitor cocktail set-I, Tris buffer, Triton X-100 and Tween-20 were from Sigma Chemicals Co. (St. Louis, MO). All other chemicals and reagents used were of highest purity commercially available. Four to six week old male Wistar rats (160–180 g), were

obtained from the animal breeding colony of CSIR-Indian Apitolisib Institute of Toxicology Research (CSIR-IITR), Lucknow, acclimatized under standard laboratory conditions and given a commercial pellet diet (Provimi Animal Nutrition India Pvt Limited, India) and water ad libitum. Animals were housed in plastic cages on rice husk as bedding and maintained in controlled atmosphere of 12 h dark/light cycle, 22 ± 2 °C temperature and 50–60% humidity as per rules laid down by Animal Welfare Committee of CSIR-IITR, Lucknow. All the experiments involving animals were approved by the Institutional Animal Ethics Committee (IAEC), CSIR-IITR, Lucknow. Animals were sacrificed by cervical dislocation with minimal suffering as per CSIR-IITR guidelines. To study the protective effect isometheptene of NX slightly modified experimental schedule of Solt and Farber liver tumorigenesis protocol was followed [14] and [15]. This modified experimental protocol eliminates partial hepatectomy (PH). Because, PH is desirable to increase the sensitivity with weak agents and PH requires extensive surgical procedure that causes a lot of pain and mortality of animals. In the classical Solt-Farber model, along with 2-AAF, PH was done for vigorous liver cell proliferation and in this protocol growth can be grossly visible within a period of 1 week.