Gemcitabine

Antibodies specific for TIE-1 (c-18) and TFIIH were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). KLF5 antibody was purchased from Abcam (Cambridge, MA, USA). Antibodies specific for XPC, cleaved-PARP, phospho-histone H2A.X (ser 139), and H2A.X were purchased from Cell Signaling Technology (Boston, MA, USA). Cc3-conjugated V5 antibody and β-actin antibody (A5441) were from Sigma (St. Louis, MO, USA). Hoechst 33342 was obtained from Dojindo (Kumamoto, Japan). Cisplatin, adriamycin, paclitaxel, 5-fluorouracil, methotrexate, carboplatin, and gemcitabine were purchased from Wako (Japan). Human TIE-1 cDNA was purchased from Kazusa DNA Research Institute (Kisarazu, Japan). Ovarian cancer cell lines were from the American Type Culture Collection. SiRNAs of TIE-1 (s14140, s14141), TIE-2 (s13983), XPC (s14929, s14930) and CSB (s 4806, s4807) are from Thermo fisher Scientific (Waltham, MA U.S.A.).

Two human invasive bladder cancer cell lines, T24 and T24PR, were used. T24 cells were obtained from the American Type Culture Collection (Rockville, MD, USA). T24PR cells were established in our laboratory as an acquired platinum resistant cell line [11 (link)]. Briefly, T24 cells were grown and passaged upon reaching confluence in medium containing CDDP over a 6-month period to develop platinum resistance. All cells were routinely maintained in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Dainippon Pharmaceutical, Tokyo, Japan), at 37°C in a humidified 5% CO₂ atmosphere. DHMEQ, synthesized as described previously [10 (link),12 (link)], was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/ml and stored at −20°C. This stock solution was diluted in culture medium to a final concentration of <0.1%. CDDP and paclitaxel were kindly supplied by Nippon Kayaku Co. (Tokyo, Japan). Gemcitabine and carboplatin were obtained from Wako Pure Chemical Industries (Osaka, Japan).

H₂O₂ and gemcitabine were purchased from Wako Pure Chemical Industries (Osaka, Japan). Rabbit antibody against p21 (#2947) were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-p53 antibody (#sc-126) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-GAPDH antibody (#2275-PC-100) was purchased from R&D Systems (Minneapolis, MN, USA). SB225002 (#S7651) and SCH-527123 (#S8506) were obtained from Selleck Chemicals (Houston, TX, USA). Other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise specified.

OVCAR‐8 cells were maintained in RPMI‐1640 medium containing 10% FBS and penicillin/streptomycin. Dasatinib was purchased from JS Research Chemicals Trading Co. (Wedel, Germany), gemcitabine from Wako Pure Chemicals (Osaka, Japan) and AZD1775 and olaparib from Adooq Bioscience (Irvine, CA, USA). For a single thymidine block, the cells were treated with 2 mm of thymidine for 24 h and released into a fresh medium with or without drugs for 12 h.

DMS 273 and NCI-H1048 cells were seeded in 96-well cell culture plates containing RPMI 1640 with various concentrations of the following inhibitors: COH29, triapine, HU, and gemcitabine (FUJIFILM Wako Pure Chemicals). After incubation at 37 °C for 72 h, cell viability was analyzed using a WST-8 assay using Cell Counting Kit-8 (Dojindo).
For the proliferation assay, DMS273 and NCI-H1048 cells were transfected with siRNA for 24 and 48 h, harvested, and Trypan Blue-negative cells were counted using a Countess automated cell counter (Thermo Fisher Scientific) to indicate the number of viable cells.