Irinotecan cpt 11

The human colorectal cancer cell lines, HCT116, HT29 and DLD-1 and lung cancer cell line 226Br were cultured in RPMI1640 (Welgene, Seoul, Korea) containing 10% fetal bovine serum (FBS; Welgene) and antibiotics (100 mg/L penicillin and 100 mg/L streptomycin; GIBCO-BRL Life Technologies; Gaithersburg, MD, USA). Human colonic fibroblasts (CCD-18co) were cultured in DMEM (Welgene, Seoul, Korea) containing 10 % FBS and antibiotics Cells were incubated in a humidified thermostat under 5 % CO₂ at 37 °C. A predesigned siRNA targeting HGF and scramble siRNA were purchased from Shanghai Genepharma (Shanghai, China). Recombinant Human HGF was purchased from R&D system (Minneapolis, MN, USA). Humanized anti-HGF antibody was kindly provided by YooYoung pharmaceutical Co. Ltd (Seoul, Korea) and iBIO Inc (Seoul, Korea). Irinotecan (CPT-11) was obtained from Sigma Aldrich (St. Louis, MO, USA). Antibodies to c-MET, phosphorylated-c-MET, phosphorylated-ERK, phosphorylated-AKT, PARP, and cleaved Caspase-3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody against HGF, actin and fibroblast marker (ER-TR7) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The human HCT116 colon carcinoma cell line was purchased from DS Pharma Biomedical Co., Ltd (Osaka, Japan). McCoy's 5A medium, Leibovitz L-15 medium (L-15), penicillin-streptomycin and fetal bovine serum (FBS) were purchased from Life Technologies (Carlsbad, CA, USA). Oxaliplatin (L-OHP), gemcitabine (Gem), DAC, AC, zebularine (Zeb), TSA and SAHA were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). 5-fluorouracil (5-FU) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Irinotecan (CPT-11), 7-ethyl-10-hydroxycamptothecin (SN-38) and VPA sodium salt were purchased from Sigma-Aldrich (St. Louis, MO, USA). Monoclonal mouse anti-β-actin (dilution, 1:1,000; catalog no. sc-4778), monoclonal mouse anti-CDA (dilution, 1:1,000; catalog no. sc-365292), goat anti-mouse immunoglobulin (Ig)G-horseradish peroxidase (HRP; dilution, 1:2,000–5,000; catalog no. sc-2005) and goat anti-rabbit IgG-HRP antibodies (dilution, 1:2,000; catalog no. sc-2004) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Monoclonal mouse anti-DNMT1 (dilution, 1:1,000; catalog no. ab-13537), polyclonal rabbit anti-ENT1 (dilution, 1:1,000; catalog no. ab-48607) and polyclonal rabbit anti-dCK (dilution, 1:500; catalog no. ab-91599) antibodies were purchased from Abcam (Cambridge, UK). All other chemicals were of the highest grade commercially available.

For immunoblotting, we obtained S6K (sc-230), PERK (sc-13073) and p-PERK (sc-32577) antibodies from Santa Cruz Biotechnology, Dallas, TX, human Sestrin2 (10795-1-AP) antibody from Proteintech, Chicago, IL, BiP (3177), CHOP (2895), p-Thr389 S6K (9234), p-Ser235/236 S6 (2211), S6 (2317), p-Thr37/46 4E-BP (2855), 4E-BP (9452), p-Ser473 AKT (9273) and AKT (4691) antibodies from Cell Signaling Technology, Danverse, MA, actin (JLA20) antibody from Developmental Studies Hybridoma Bank (DSHB, Iowa city, IA), and tubulin (T5168) antibody from Sigma, St. Louis, MO. Mouse Sestrin2 antibody was described (Ro et al, 2014b (link)). For immunostaining, we obtained p53 (sc-6243), PCNA (sc-7907), β-catenin (sc-59737), CHOP (sc-575) from Santa Cruz Biotechnology, F4/80 (mf48000) from Invitrogen, Carlsbad, CA, BiP (3177), γ-H2AX (2577), p-Ser235/236 S6 (2211) and p-Thr37/46 4E-BP (2855) from Cell Signaling Technology. Azoxymethane (AOM), dextran sulfate sodium (DSS), rapamycin, 5-fluorouracil (5-FU) and irinotecan (CPT-11) were from Sigma.