Tcpobop

Compound 1 (CITCO, (6-(4-chlorophenyl)imidazo [2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichloro-benzyl)oxime)),
rifampicin (rif), TCPOBOP, and PK11195 were obtained from Sigma-Aldrich
(St. Louis, Missouri, United States, now Merck), which is now known
as Merck (Darmstadt, Germany). Phenobarbital (Luminal 200 mg/mL injection)
was manufactured by Desitin Pharma spol. s.r.o. (Prague, Czech Republic).
Ligands for nuclear receptors (GW3965, thyroxin, obeticholic acid,
dexamethasone, fenofibrate, GW501516, rosiglitazone, 3-methylcholanthrene,
calcitriol, testosterone, and estradiol) were purchased from Sigma-Aldrich
(now Merck). The prototype ligands were used at 100 nM (dexamethasone,
calcitriol), 1 μM (thyroxin), or 10 μM concentrations.
The compounds were dissolved in DMSO, and the final concentration
of DMSO in the entire reaction mixture or cultivation media was 0.1%.

A two-step strategy was used for hepatocarcinogenesis [23 (link)]. Briefly, 14–15 day old mice were injected with DEN (20 mg/kg body weight) (Sigma) to initiate tumor growth. Beginning at day 28, mice were injected with a phenobarbital-like inducer TCPOBOP (3 mg/kg body weight) (Sigma) once every two weeks for a total of eight times to promote tumor growth. Subsequently, these mice were randomly assigned into two groups. Early Group were treated with either adenovirus for Dlk1 knockdown or adenovirus for luciferase knockdown as control (5 × 10⁹ particle per animal) intravenously every 3 weeks for a total of 4 times, starting 4 weeks after DEN administration, while Late Group were injected with that for a total of 5 times, starting 16 weeks after DEN injection. Finally the mice were sacrificed to evaluate the effect of DLK1 knockdown on hepatocarcinogenesis by examining tumor number, size and weight.

Paclitaxel, CITCO, TCPOBOP, androstenol and MTT were obtained from Sigma-Aldrich (St. Louis, MO, USA). Media and reagents for cell culture were acquired from Invitrogen (Carlsbad, CA, USA). Trizol, oligoDT primers, Superscript II enzyme and the Power SYBR Green mastermix were from Life Technologies (Grand Island, NY, USA). Other reagents were of analytical grade. The cell lines used in this experiment were the mouse cell line E9 [21] (link) and the human cell lines A549, H2023, H460, H2030, H1792 and H23 [22] (link). All these cell lines were a gift from Dr. Lucy M. Anderson from the Laboratory of Comparative Carcinogenesis at the Frederick National Laboratory for Cancer Research (United States of America).

Human liver cancer cell lines including PLC/PRF/5, QGY7703, Huh7, etc., normal liver cell line Chang and mouse liver cancer cell line Hepa1-6 were all purchased from Cell Bank of the Typical Culture Preservation Committee, Chinese Academy of Sciences (Shanghai, China). PLC/PRF/5, Huh7,Hepa1-6, HepG2, SK-Hep1 and HCCLM3 cells were cultured in the DMEM medium (Invitrogen, Shanghai, China).And Chang, QGY7703, BEL7402 were cultured in RPMI1640 medium (Invitrogen, Shanghai, China). All mediums were supplemented with 10% FBS and 100U/mL penicillin–streptomycin. The following antibodies were used for Western blotting: WIP1 (sc-376257) to detect human samples from Santa Cruz (Shanghai, China); WIP1 (A6204) to detect mouse samples from ABcolonal (Wuhan, China); cleaved PARP1(#9541), cleaved-caspase3 (#9661) and beta-Actin (#4970) from Cell Signaling Technology (Shanghai, China); γH2AX (phospho-Histone H2AX (s139)) (ab81299) from Abcam (Shanghai, China); ki67 (ER1802-31) from Huabio (Hangzhou, China). WIP1 plasmid was kindly provided by Prof. Zhenyu Ju at Hangzhou Normal University. GSK2830371, Diethylnitrosamine (DEN) and TCPOBOP were purchased from Sigma-Aldrich (Shanghai, China). Olaparib (HY-10162) and Veliparib (HY-10129) were purchased from MedChemExpress (Shanghai, China). Other reagents and chemicals don’t list here are commercially available.

The proteasome inhibitor MG132 was purchased from Calbiochem (San Diego, CA), and TCPOBOP was purchased from Sigma (St. Louis, MO). Dual Luciferase kits were purchased from Promega Corp. (Madison, WI). Minimal essential media and L-glutamine were purchased from Invitrogen Corp. (Carlsbad, CA), and penicillin/streptomycin was purchased from Sigma. Fugene transfection reagent was purchased from Roche Diagnostics GmbH (Indianapolis, IN), and Lipofectamine 2000 was purchased from Invitrogen.