Fli 06

To infuse the protein solution, the mice were anesthetized with 3% isoflurane for induction and 1.5% isoflurane for maintenance by using an inhalant anesthesia machine (RWD Life Science). The intracranial cannula was then connected to a 10 μL microsyringe using a polyethylene tube, and 5 μL of the solution (2 μg/mL) was administered at a flow rate of 0.5 μL/min using a microsyringe pump. The control group was administered the same volume of normal saline. After infusion, the microsyringe remained in situ for an additional 5 min. The internal injection cannula was then slowly pulled out, and the stylet was placed back into the guide cannula. A protein solution or normal saline was injected daily for 10 consecutive days. To demonstrate the dose-dependent effects of CCN3 (R&D System, USA, 1976-NV-050), cells were treated with a series of recombinant mouse CCN3 protein concentrations (5, 10, 20, 50, 100, 200, and 500 ng/mL) for 3 days. FLI-06 (Sigma-Aldrich, 20 Μm, SML0975) or VO-OHpic (Tocris, Bristol, UK, 5 μM, 3591) was used to block the effects of CCN3 or shCCN3 for 3 days, with equivalent DMSO used as vehicle control. All in vitro treatments were performed in at least three independently prepared NSC cultures.

DMBA, TCDD, and alpha-naphthoflavone (α-NF) were purchased from Toronto Laboratories Research (Toronto, Canada). Dulbecco’s Modified Eagle’s Medium (DMEM), TRIzol reagent, Annexin V-FITC/Propidium Iodide (PI) were purchased from Invitrogen Co. (Grand Island, NY). FLI 06, LY294002, sodium azide, and puromycin were purchased from Sigma-Aldrich Co., (St. Louis, MO), whereas XAV-939 was obtained from Tocris Bioscience (Bristol, UK). High Capacity cDNA Reverse Transcription kit and SYBR® Green PCR Master Mix were purchased from Applied Biosystems® (Foster city, CA). DNA primers were obtained from Integrated DNA Technologies (Coralville, IA). Aldefluor^® kit was purchased from Stem Cell Technologies (Vancouver, CANADA). Acrylamide/bisacrylamide 30% (29:1) and nitrocellulose membrane were purchased from Bio-Rad Laboratories (Hercules, CA). Vybrant apoptosis assay kit Molecular Probes® were obtained from Thermo Fisher Scientific (Waltham, MA). Primary and Horse Radish Peroxidase (HRP)-conjugated secondary antibodies against target proteins, AhR and CYP1A1 shRNA, and transfection reagent systems were ordered from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The Western blot detection kits, Luminata® Western HRP Chemiluminescence Substrates were purchased from EMD Millipore (Billerica, MA).

Human embryonic kidney 293 cells T cells (ATCC CRL-3216) were maintained in DMEM supplemented as above. All cells were grown at 37°C and 5% CO₂.
Where indicated, cells were treated with 10 - 500 ng/ml doxycycline (dox), 10 μg/ml brefeldin A (BFA, B8500, LC laboratories) and 10 μM FLI-06 (SML0975, Sigma). All the cell lines generated in this study are indicated in Table S2 and Key Resources Table. All experiments were performed at cell densities of 70%–90% confluence.