Caspase 8

DMEM medium, penicillin, streptomycin, collagenase, PMA, Fumonisin B-1 (FB-1), Imipramine and TRI Reagent were purchased from Sigma (St Louis, MO). Fetal calf serum was purchased from Gibco BRL (Grand Island, NY). Deoxynucleoside triphosphates, RevertAid M-MuLV Reverse Transcriptase, oligodT, RNase inhibitor and other chemicals for complementary DNA synthesis were from Fermentas (Ontario, Canada). Anti- PKCα, PKCβ, PKCδ, PKCθ, PKCε, PKCζ, PLD1, pAKT, AKT, procaspase 3 caspase 3, procaspase 8, caspase 8, Bax and Bcl-2 antibodies were purchased from Santa Cruz Biotechnology (San Jose, CA). PKCα, PKCδ, PLD1 small-interfering RNA (siRNA) and control siRNA were obtained from Santa Cruz Biotechnology.

Tissue proteins were extracted and their concentration was determined using a BCA kit (Wuhan Boster Biological Technology Ltd., Wuhan, Hubei, China). The protein extracts were boiled in a sample buffer for 10 min, separated on a 10% polyacrylamide gel, and transferred to PVDF membrane. The membranes were blocked with 5% bovine serum albumin (1 h, RT), and incubated with primary antibodies (Cell Signaling Technology) for Wnt3 (1:1000 dilution, #9248), GSK-3β (1:1000 dilution, #8240), glyceraldehyde phosphate dehydrogenase (GAPDH) (1:500 dilution, #5174), and β-catenin (1:1000 dilution, #9566). In addition, antibodies against TCF-1 (1:1000 dilution, sc-79542), Bax (1:1000 dilution, sc-23959), Bcl-2 (1:1000 dilution, sc-7382), caspase-3 (1:1000 dilution, sc-65496), and caspase-8 (1:1000 dilution, sc-81656) purchased from Santa Cruz Biotechnology were used. The membranes were then washed in TBST and incubated with corresponding secondary antibodies (1 h, RT). GAPDH was used as an internal reference. Bio-Rad Gel DOC EZ imager was used to develop the membranes. The images were evaluated using the Image J software.

On a small-scale, extract of Cedrus atlantica (CAt) from Morocco was prepared in our laboratory. The bark of CAt (600g) was extracted by steam-distillation with a flow rate of approximately 7.2 ml/min at 100~105℃ for 90 mins. On a large-scale, preparation of CAt extract was commissioned to Phoenix (New Jersey, USA) following the above conditions. CAt extract and temozolomide (purity ≥ 99%, International Laboratory USA, CA, USA) were dissolved in dimethyl sulfoxide (DMSO) before each experiment, and a final concentration of DMSO was < 0.5% in experiments of cells treatment.
An antibody against p-H2A.X was purchased from Cell Signaling Technology (Beverly, MA, USA), p-ATR, p-Chk2 were purchased from Biorbyt Ltd. (Cambridge, UK), and β-actin were purchased from iReal Biotechnology (Hsinchu, Taiwan). Antibodies used to detect p-ATM, p-Chk1, p53, p-p53, p21, p-RB CDK4, cyclin D1, CDK2, cyclin A, cyclin B1, PCNA, Fas, Fas-L, Bax, Bcl-2, AIF, caspase-8, caspase-9, caspase-3, AKT, p-AKT, mTOR, p-mTOR, P70S6K and p-P70S6K were purchased from Santa Cruz Biotechnology (CA, USA). The detail information of antibodies were descripted in Table 1.

Quantitative RT-PCR (qRT-PCR) assay was accomplished with Trizol (Invitrogen, Carlsbad, CA, USA) for total RNA isolation, ReverTra Ace qPCR RT Kit (Toyobo, Japan) for reverse transcription and SYBR Green Realtime PCR Master Mix (Toyobo, Japan) for quantitation, according to the corresponding protocols. mRNA expression levels of MRP1, survivin and β-catenin were evaluated by their specified primers (Supplementary Table S1) with GAPDH as an internal control. Each assay was done in triplicate.
Primary antibodies against Nanog and survivin (Cell Signaling Technology, Danvers, MA, USA), caspase 8, caspase 3, PARP and TRAIL (Santa Cruz biotechnology, Santa Cruz, CA, USA) along with GAPDH (CoWin Bioscience, Bejing, China) were used for western blot. All the secondary antibodies were purchased from Santa Cruz biotechnology. CAR expression level were detected by fluorescence-activated cell sorting with PE-conjugated primary antibody against CAR (Millpore, Billerica, MA, USA) and mouse IgG-1 as isotype control (BD, Franklin Lakes, NJ, USA)

Apoptotic proteins have central role in regulation of programmed cell death via inducing (pro-apoptotic) or inhibiting (anti-apoptotic) apoptosis. 1 × 10⁶ cells/mL were treated with NTC and standard (5-fluorouracil) separately for 24 h. One milliliter of cells were then aspirated, lysed in 300 μl of Tris-HCL buffer and resolved on 10% SDS-polyacrylamide gels followed by transferring of the proteins to PVDF membranes (Millipore) and blocking with 5% non-fat dry milk in PBS- T (0.05% Tween 20) for 1 h at room temperature. Primary antibodies including Bid (sc-11243, 1:1000), caspase-3 (sc-7148, 1:1000), caspase-8 (sc-56070, 1:1000), caspase-9 (sc-56076, 1:1000), anti β-actin (sc-47778, 1:5000), Bax (sc-23959, 1:1000) Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), Bcl-X_L (ab32370, 1:1000), Bcl-2 (ab7973, 1:1000), and p53 (ab2433, 1:1000) (Abcam Inc., Cambridge, MA, USA). Secondary antibodies conjugated to horseradish peroxidase were obtained from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, MD, USA). Protein-antibody complexes were detected using Amersham ECL prime Western blotting detection reagent (GE Healthcare, Munich, Germany).