Accelerated degradation of cFLIPL and sensitization of the TRAIL DISC-mediated apoptotic cascade by pinoresinol, a lignan isolated from Rubia philippinensis

Plant-derived lignans have numerous biological effects including anti-tumor and anti-inflammatory activities. Screening of purified constituents of Rubia philippinensis from human glioblastoma cells resistant to TNF-related apoptosis-inducing ligand (TRAIL) has suggested that the lignan pinoresinol was a highly active TRAIL sensitizer. Here we show that treatment with nontoxic doses of pinoresinol in combination with TRAIL induced rapid apoptosis and caspase activation in many types of glioblastoma cells, but not in normal astrocytes. Analyses of apoptotic signaling events revealed that pinoresinol enhanced the formation of TRAIL-mediated death-inducing signaling complex (DISC) and complete processing of procaspase-8 within the DISC in glioblastoma cells, in which caspase-8 was inactivated. Mechanistically, pinoresinol downregulated the expression of cellular FLICE-inhibitory protein (cFLIPL) and survivin through proteasome-mediated degradation, without affecting death receptors or downstream intracellular apoptosis-related proteins. Furthermore, the sensitization of TRAIL-mediated apoptosis by pinoresinol strictly depended on the expression level of cFLIPL, which was regulated through de novo protein synthesis, rather than by NF-κB or p53 signaling. Taken together, our results indicate that pinoresinol facilitates DISC-mediated caspase-8 activation by targeting cFLIPL in an early event in apoptotic signaling, which provides a potential therapeutic module for TRAIL-based chemotherapy.

The use of TNF-related apoptosis-inducing ligand (TRAIL) in cancer therapy has long been thought as an attractive strategy because it can selectively target cancer cells without affecting the majority of normal human cells 1 . The anti-cancer activity of TRAIL is attributable to its ability to elicit apoptosis through binding of its functional receptors, death receptors 4 and 5 (DR4 and DR5), and subsequent association with an adaptor protein, Fas-associated death domain (FADD) 2 . During apoptosis, FADD recruits the initiator caspases (procaspase-8 and/or procaspase-10) for the assembly of a death-inducing signaling complex (DISC). Within the DISC, the oligomerization and cleavage of the initiator caspases are the critical upstream events for activation of either the executioner caspase-3 cascade or mitochondrial-dependent apoptotic pathway via Bid cleavage, leading to sufficient apoptosis upon TRAIL treatment, depending on the specific cell type 3,4 . Importantly, genetic lesions in the components of TRAIL signaling have been found in human malignant cancers, suggesting that TRAIL functions in immune surveillance against developing cancers and metastasis [5][6][7] . Indeed, mice null of TRAIL receptor were shown to susceptible to inflammation and tumorigenesis with apoptotic defects 8 . Consistent with this possibility, currently evaluated TRAIL agonistic antibodies have demonstrated a significant therapeutic efficacy in a number of preclinical studies 9,10 . However, therapeutic benefits in clinical trials have been rather limited due to the Results IIdentification of pinoresinol from R. philippinensis as a tRAiL sensitizer in tRAiL resistant glioma cells. We characterized a set of major compounds obtained from R. philippinenesis to identify active constituents that synergistically sensitized the cytotoxic effects of TRAIL in TRAIL-resistant glioblastoma cells (Supplementary Table S1, Supplementary Figs 1-33). Treatment of LN428 cells with 50-200 ng/mL TRAIL alone induced a limited number of cell deaths (<5%) over 24 h (data not shown). In the screening assay, LN428 cells were sequentially treated with the purified compounds and 50 ng/mL TRAIL, followed by an ATP-based cell viability assay. In parallel, we tested the cytotoxicty of each compound on LN428 cells as single agents. Of the compounds screened, the lignin pinoresinol was a potent sensitizer of TRAIL-mediated cytotoxicity (Fig. 1A,B). It eliminated the survival of LN428 cells but only in the presence of TRAIL; it had only marginal growth inhibitory effects as a single agent (Fig. 1C). Consistently, no cell death was observed when cells were treated with pinoresinol alone at concentrations up to 1 μM over 24 h. By contrast, combined treatment with the same concentrations of pinoresinol and TRAIL resulted in a drastic increase in cell death (Fig. 1D), thus confirming that this combination resulted in extensive cell death at low concentrations (0.2-1 μM) of pinoresinol.  Sensitization of TRAIL-induced apoptosis by non-toxic dose of pinoresinol requires caspase-8 activation in various glioblastoma cells, but not in normal astrocytes. (A) LN428 cells were treated with PINO (0.5 μM), TRAIL (50 ng/ml) and TRAIL (50 ng/ml) plus PINO (0.5 μM) for the indicated times. (B) TRAIL resistant glioblastoma cells (U87MG, LNZ308, LN428, and U251) and normal primary astrocytes were treated with PINO, TRAIL and TRAIL plus PINO for 24 h. Cell death was quantified as in Fig. 1A. Data were normalized to the rate of spontaneous cell death occurring in untreated cells. Data represents the mean ± SE of three independent experiments. *p < 0.05, compared with the TRAIL only-treated group. (C,D) LN428 cells were treated with PINO, TRAIL and TRAIL plus PINO for 24 h in the absence or presence of caspase or necroptosis inhibitor z-VAD-fmk (20 μM)/z-IETD-fmk (50 μM) or Nec-1 (30 μM). (C) Cell death was quantified as in A. Data represents the mean ± SE of three independent experiments. *p < 0.05, compared with the PINO/TRAIL-treated group. (D) Cells were visualized using an inverted microscope. (E) LN428 cells were treated with PINO, TRAIL and TRAIL plus PINO for 24 h in the absence or presence of z-VAD-fmk. Cells were subjected to Annexin V/PI staining, and then analyzed by flow cytometry. (F) LN428 cells were treated with PINO (0.5 μM), TRAIL (50 ng/ml) and TRAIL plus PINO for indicated times. Whole cell lysates were subjected to immunoblotting with the indicated antibodies (left) and densitometry analysis of the bands from the relevant proteins was performed (right). Data represents the mean ± SE of three independent experiments. *p < 0.05, compared with the TRAIL only-treated group. be limited to cancer cells. Although TRAIL-induced cancer cell death was mainly apoptotic, it might also induce non-apoptotic cell death via non-canonical TRAIL signaling depending on the cellular context [19][20][21] . Thus, next, we examined whether cell death caused by pinoresinol plus TRAIL was associated with caspase-dependent apoptosis. As expected, pretreatment of LN428 cells with pancaspase and the irreversible caspase 8-inhibitors z-VAD-FMK and z-IETD-fmk completely abrogated the cytotoxicity induced by pinoresinol plus TRAIL (Fig. 2C,D). However, necrostain-1, an inhibitor of programmed necrosis, failed to protect against cell death, indicating that pinoresinol predominantly triggered apoptotic, rather than necrotic, cell death. To confirm the mode of TRAIL-mediated cell death sensitized by pinoresinol, cell death was analyzed by Annexin V and propidium iodide (PI) staining followed by flow cytometry. Consistently, treatment of pinoresinol plus TRAIL drastically increased the population of an early phase of apoptosis (Annexin V + ), whereas very few cells were stained exclusively with PI, and such increased apoptotic population was prevented by co-treatment with z-VAD-FMK (Fig. 2E). To get more insights into the mechanisms underlying TRAIL-sensitized apoptosis, we sequentially analyzed the activation of processes of caspase signaling cascade, including those of initiator caspase (caspase-8) and as executor caspases (caspase-3 or −9) and the resultant PARP cleavage. In the kinetic analysis, we found that the treatment of pinoresinol plus TRAIL caused an activation of caspase-8 and −3, and PARP cleavage from 6 h onwards (Fig. 2F). Furthermore, pretreatment with z-IETD-fmk completely inhibited the activation of caspase cascade induced by pinoresinol plus TRAIL treatment. These results clearly indicate that casapse-8 activation is essential in the sensitization of pinoresinol-induced apoptosis in TRAIL-resistant glioblastoma cells.
Sensitizing efficacy of pinoresinol on TRAIL-mediated apoptosis is not associated with either nf-κB or p53. Pinoresinol exhibits anti-inflammatory properties via blockade of the NF-κB pathway in several immune and cancer cells [22][23][24] . Given the well-established ability of NF-κB to regulate TRAIL resistance through induction of its anti-apoptotic genes 25 , it was hypothesized that the anti-NF-κB effects of pinoresinol might contribute to sensitization against TRAIL-induced apoptosis. Consistent with previous studies [22][23][24] , pretreatment of LN428 cells with pinoresinol significantly decreased the transcriptional activity of NF-κB induced by either TNF or TRAIL, while pinoresinol alone did not affect the basal level of NF-κB activity (Fig. 3A). However, unexpectedly, LN428 cells with prevention of NF-κB activation by overexpression of the IκBα super-repressor (SR-IκBα), which could not be phosphorylated due to substitutions of serine 32 and serine 36 by alanine, were found to have a similar extent of cell death after TRAIL or pinoresinol plus TRAIL treatment, although TNF-induced cell death was drastically enhanced (Fig. 3B). In addition, pretreatment with the NF-κB inhibitor TPCA failed to affect cell death upon TRAIL or pinoresinol plus TRAIL treatment (Fig. 3C). These results suggest that the TRAIL-sensitizing efficacy conferred by pinoresinol was unlikely to be a result of NF-κB inhibition. Although p53 activation plays an important role in TRAIL sensitization 1,26 , the involvement of p53 in pinoresinol-induced TRAIL sensitization was excluded because LN428 cells retained mutant-p53 27 . Consistent with this possibility, we found that pinoresinol treatment also sensitized TRAIL-induced cell death in p53 null HCT116 cells to a similar extent as it did with wild type (WT) cells, despite the remarkable differences in cytotoxicity after camptothecin treatment between these two cell types (Fig. 3D). Moreover, no detectable induction of p53 and its target p21 upon pinoresinol alone or pinoresinol plus TRAIL treatment in WT-HCT116 cells, despite the fact that the substantial amounts of p53 and p21 were induced by a DNA damaging agent, camptothecin in WT-HCT116 cells but not in p53-null HCT116 cells (Fig. 3E). Such findings thus indicate that pinoresinol's effect on TRAIL-mediated cell death is independent of p53. pinoresinol accelerates DiSc formation by down-regulating cfLip L expression. To characterize the underlying mechanism involved in pinoresinol-induced sensitization of glioma cells against TRAIL-mediated apoptosis, we determined the expression levels of several apoptosis-related proteins in the death receptor signaling pathway after exposure to pinoresinol for different times in LN428 cells. Notably, the protein expression levels of the long isoform of cellular FLIP (cFLIP L ) and survivin were drastically decreased in a time-dependent manner in LN428 cells with pinoresinol ( Fig. 4A, panels 6,7). Reductions of cFLIP L and survivin were accompanied by increased levels of cleaved-RIP1 and truncated-Bid (t-Bid) in cells upon pinoresinol plus TRAIL treatment (Fig. 4A, panels 4,11). Furthermore, we observed that pinoresinol was also able to down-regulate cFLIP S expression with a similar kinetics with cFLIP L in HT-29 cells, though the expression level of cFLIP S was extremely low or nondetectable in glioblastoma cells including LN428 cells and LNZ308 cells (Fig. 4B). These results suggest that expression of cFLIP isoforms is highly cell type-specific and pinoresinol-induced cFLIP downregulation, especially in cFLIP L , may play a predominant role in sensitizing TRAIL-mediated apoptosis in glioblastoma cells. By contrast, the protein levels of signaling components of TRAIL including death receptors, adaptor proteins, other inhibitor of apoptosis proteins, and Bcl-2 family proteins were not affected or only modestly affected in cells after pinoresinol treatment. The expression of cFLIP L and survivin proteins are regulated by either transcriptional or post-translational modifications such as ubiquitin-mediated proteasomal degradation [28][29][30][31] . In contrast to the observed down-regulation of cFLIP L and survivin protein levels, treatment with pinoresinol did not change their mRNA levels at any of the time points examined (Fig. 4C). However, pretreatment with the proteasome inhibitor MG132 sufficiently prevented the down-regulation of cFLIP L and survivin expression by pinoresinol (Fig. 4D), suggesting that pinoresinol might reduce the protein levels of cFLIP L and survivin via proteasome-mediated degradation rather than through transcriptional control.
Next, we examined whether downregulation of cFLIP L and survivin by pinoresinol affected the facilitated TRAIL-mediated cytotoxicity by overexpressing these genes in LN428 cells. Consistent with its critical function to antagonize caspase-8, overexpression of WT cFLIP L resulted in a significant decrease in cell death and caspase cascade activation induced by pinoresinol plus TRAIL treatment (Fig. 5A,B). We also found that a cFLIP L mutant (cFLIP L -K167/195R containing modified major ubiquitin acceptor sites) more profoundly abrogated pinoresinol plus TRAIL-induced caspase-dependent apoptosis, compared to that of WT cFLIP L . However, the cell death www.nature.com/scientificreports www.nature.com/scientificreports/ induced by pinoresinol plus TRAIL was not affected by the overexpression of survivin, suggesting that downregulation of upstream anti-apoptotic protein anti-cFLIP L , rather than survivin, contributed to an important mechanism involving pinoresinol sensitization of TRAIL-induced cell death.
The cFLIP isoforms compete directly with procaspase-8 for binding to FADD in a TRAIL-dependent fashion, thus inhibiting procaspase-8/-10 recruitment to form the DISC 31,32 . It is therefore possible that downregulation of cFLIP L by pinoresinol might directly affect the formation of the DISC, an early signaling event in TRAIL-induced apoptosis. An immunoprecipitation assay using an anti-caspase-8 antibody revealed that treatment of LN428 cells with TRAIL in the absence of pinoresinol led to efficient recruitment of cleaved cFLIP L to the isolated DISC, whereas DISC-bound FADD and caspase-8/-10 were only weakly detected (Fig. 5C, left panel, rows 1-3). These results suggest that in LN428 cells, the TRAIL-induced recruitment of cFLIP L into the DISC was an important step before caspase-8 activation to exhibit resistance against TRAIL cytotoxicity. However, pretreatment with pinoresinol promoted an increase in TRAIL-mediated DISC formation and procaspase-8/10 processing, concomitant with decreasing amounts of DISC-bound cFLIP L (Fig. 5C, left panel, rows 4-6). More importantly, in pinoresinol-pretreated cells, activation of procaspase-8 processing within the DISC following TRAIL treatment proceeded to completion, as shown by the appearance of the active p18 subunit of mature caspase-8. Taken together, these results strongly suggest that a reduced amount of DISC-bound cFLIP L played a major role in TRAIL sensitization by pinoresinol.
pinoresinol-mediated down-regulation of cfLip L is mediated via de novo protein synthesis inhibition. Next, we identified the underlying mechanism by which pinoresinol directly controls ubiquitin-mediated degradation of cFLIP L. As expected, co-immunoprecipitation analyses showed that treatment of cells with MG132 led to an increase in polyubiquitinated cFLIP L with concomitant enhanced protein levels (Fig. 6A). However, we unexpectedly detected lower levels of ubiquitinated cFLIP L in cells treated with pinoresinol plus MG132, compared to cells exposed to MG132 cells, indicating that the accelerated proteasomal degradation of cFLIP L by pinoresinol was not achieved through direct activation of the ubiquitination process. www.nature.com/scientificreports www.nature.com/scientificreports/ Given that cFLIP L and survivin are unstable proteins with a rapid turnover 29,33 , we addressed whether the reduced protein levels by pinoresinol were associated with de novo protein synthesis of cFLIP L and survivin. Treatment with either pinoresinol or cycloheximide (CHX) did not influence the cellular amounts of DRs and adaptor proteins, including DR4/5, FADD, RIP1, and TRAF2 (Fig. 6B). By contrast, pinoresinol was able to down-regulate the expression levels of cFLIP L and survivin with similar kinetics to that of CHX. Furthermore, the down-regulating effect by either pinoresinol or CHX was not accelerated by the combined treatment of pinoresinol and CHX. These results suggest that in a similar manner to CHX, pinoresinol inhibited de novo synthesis of proteins with a rapid turnover cFLIP and survivin. www.nature.com/scientificreports www.nature.com/scientificreports/ To directly assess whether the down-regulation of protein expression by pinoresinol is due to the impairment of the general translational machinery, we conducted a cell-free in vitro transcription and translation assay. As shown in Fig. 6C, pinoresinol suppressed the production of green fluorescent protein (GFP), similar to a well-known protein translation inhibitor CHX, in a dosage dependent manner. To rule out the possibility that pinoresinol-dependent suppression of protein synthesis is caused by hampering transcriptional processes, subsequent in vitro translation response was assessed by using in vitro synthesized EGFP mRNA. Consistently, incubation with 1 μM pinoresinol completely interfered the EGFP protein production with a similar efficacy of 10 μM CHX (Fig. 6D). Taken together, these data indicate that pinoresinol directly interferes a de novo protein synthesis without affecting transcriptional machinery.

Discussion
Glioblastoma is a heterogenous group of invasive malignant primary brain tumors with high mortality 34 . Although all populations of cancer cells contribute in their own way to drive tumor growth, the molecular changes disrupting the apoptotic pathway are considered a pathological hallmark of glioblastoma. Emerging evidence suggests that cells within the glioblastoma exhibit abnormalities of the cell death pathway such as overexpression of antiapoptotic proteins or silencing of key death effectors 35,36 . Importantly, genomic analyses of human glioblastomas have shown that caspase-8, an essential component of the DISC, is frequently inactivated by either gene mutations or promotor methylation 7,37-39 . Thus, resistance to DR-mediated cytotoxicty in glioblastoma cells might occur as a step of DISC assembly. Consistent with this possibility, we found that in a series of glioblastoma cells including LN428 cells treated with TRAIL, complete activation of caspase-8 and functional DISC formation were blocked. However, pinoresinol treatment resulted in cells resistant to apoptosis with an  Fig. 1A. Data were normalized to the rate of spontaneous cell death occurring in untreated cells. Data represents the mean ± SE of three independent experiments. *p < 0.05, compared with the mocktransfected group. # p < 0.05, compared with the wild-type FLIP L -transfected group. (B) Whole cell extracts were subjected to immunoblotting with the indicated antibodies (left), and densitometry analysis of the bands from the relevant proteins was performed (right). Data represents the mean ± SE of three independent experiments. *p < 0.05, compared with mock-transfected group. (C) LN428 cells were treated with TRAIL (50 ng/ml) in the absence or presence of PINO (0.5 μM) for the indicated times. Cell extracts from each sample were subjected to immunoprecipitation with anti-caspase-8 antibody. Immunoprecipitants were analyzed by immunoblotting with indicated antibodies. A total of 1% of the cell extract volume from each sample was used as input control (left). Densitometry analysis of the bands from the relevant proteins was performed (right). Data represents the mean ± SE of three independent experiments. *p < 0.05, compared with none-treated group. www.nature.com/scientificreports www.nature.com/scientificreports/ increased recruitment of both procaspase-8 and FADD to the TRAIL DISC, and complete activation of caspase-8. It is therefore possible that pinoresinol-induced TRAIL sensitization was conducted at the level of the DISC/ caspase-8 axis.
Accumulating evidence presently suggests that cFLIP is a key player in the DR-mediated apoptotic pathway that retains the sublethal activation of caspase-8 at the DISC 31,32,40 . Consequently, elevated levels of cFLIP in tumor tissues from patients with a variety of cancers including lung cancer, Burkitt's lymphoma, cervical caricinoma and colorectal carcinoma are correlated with poor clinical outcomes [41][42][43][44] , implicating the existence of a strong association between suppression of DISC-mediated apoptosis by cFLIP and tumorigenesis. An important finding from the present study is that protein levels of cFLIP L were significantly reduced by pinoresinol treatment, and the ectopic overexpression of cFLIP L , significantly suppressed caspase-8 activation and reduced the susceptibility to cell death caused by pinoresinol/TRAIL treatment in LN428 cells. Changes in the expression levels of cFLIP L by pinoresinol therefore appear to be responsible for TRAIL sensitization in glioma cells. In this regard, it is important to determine the potential mechanism involved in the pinoresinol-induced downregulation of www.nature.com/scientificreports www.nature.com/scientificreports/ cFLIP L expression. It has previously been reported that cFLIP L expression is tightly regulated at the transcriptional level by a number of stimuli, including NF-κB transcription factor 45,46 , mitogen-activated protein kinase 47 and protein kinase B/Akt 48,49 . Previous pharmacological and biochemical studies have reported that pinoresinol exhibits anti-inflammatory and anti-cancer effects, in part through the inhibition of NF-κB [22][23][24] . It is therefore possible that pinoresinol suppresses cFLIP L expression through NF-κB inhibition. In accordance with previous observations, we found that pinoresinol potently inhibited the NF-κB activity in LN428 cells in response to TNF and TRAIL. However, we did not observe a decrease in cFLIP L mRNA expression levels in cells treated with pinoresinol concentrations of 0.5-1 μM, which caused cFLIP L depletion and maximal TRAIL sensitization. Furthermore, the expressions of a subset of NF-κB-inducible genes, including TRAF2, cIAP1/2, XIAP, and Bcl-X L , were unaffected by pinoresinol treatment. These findings raise the possibility that down-regulatory effects of pinoresinol on cFLIP L expression are not associated with transcriptional regulation of NF-κB. These discrepancies of transcriptional regulation between cFLIP L expression and NF-κB may have resulted from different concentrations of pinoresinol. Indeed, pinoresinol must be used at a relatively high concentration (≥10 μM) to exhibit anti-NF-κB activity in several types of cells 22,23,50 , suggesting that other factors may be involved in the effects of cFLIP L depletion by low concentrations of pinoresinol.
On the other hand, the expression levels of cFLIP L were regulated by the ubiquitin-proteasomal pathway with a short half-life 29,51 . In this respect, it is of particular interest that, in LN428 cells, pinoresinol induces proteasomal degradation of cFLIP L or ectopic expression of the cFLIP L mutant (cFLIP L -K167/195 R), which significantly abolishes sensitization by pinoresinol to TRAIL-induced cytotoxicity. Furthermore, the results of an in vitro translational assay showed that pinoresinol directly inhibited de novo protein synthesis, which has similar efficiency with the well-known protein synthesis inhibitor CHX. These results raise the possibility that pinoresinol disrupts de novo protein synthesis, particularly for fast turnover proteins such as cFLIP L , leading to proteasomal degradation with decreased stability. Nevertheless, it is currently unclear how pinoresinol inhibits de novo protein synthesis. Further studies to identify the ribosomal proteins that interact with pinoresinol in the translational machinery will be critical for a complete understanding of the mechanism of action, and for the development of novel TRAIL-based chemotherapies. Earlier, it has been reported that survivin plays an essential role in cell cycle progression 52 . In this study, we found that pinoresinol induced a G2/M arrest with an increase in the G2 population ( Supplementary Fig. 34), suggesting that down regulation of survivin by pinoresinol might be relevant in limiting cell division, especially in G2-M phase rather than apoptosis. In addition, it has recently been reported that pinoresinol suppresses the efflux of chemotherapeutic drugs outside by interacting with P-glycoprotein (P-gp) encoded by multidrug resistant-1 (MDR-1) gene 53,54 . Since P-gp efflux function was shown to contribute to TRAIL resistance via controlling the endogenous level of TRAIL in certain types of cancer cells 55,56 , such an anti-Pgp activity of pinoresinol might constitute another mechanism in enhancing TRAIL efficacy in TRAIL-resistant cancers including glioblastoma. Thus, future bioavailability study of TRAIL and pinoresinol using in vivo preclinical models are required to further validate TRAIL and pinoresinol-based therapeutic development for glioblastoma. Methods isolation of pinoresinol. Pinoresinol was isolated from our chemical study on Rubia philippinensis, as described previously 17 . Briefly, a methylene chloride (CH 2 Cl 2 )-soluble fraction (50 g) was prepared from solvent extraction of R. philippinensis extract (150 g) using CH 2 Cl 2 and water. Vacuum liquid chromatography of CH 2 Cl 2soluble fraction on silica gel column (20 × 20 cm) eluting with n-hexanes-EtOAc (20:1, 10:1, 5:1, 3:1, 2:1) and cell culture and primary astrocytes preparation. Human malignant glioblastoma cells (LN428, LNZ308, U87MG, and U251MG) were kindly provided by Dr. Yongwan Kim (Dongsung Cancer Center, Daegue, Korea). HCT 116, a human colorectal cancer cells and its p53-knockout derivates were kindly provided by Dr. Deug Y. Shin (Dankook University, Cheonan, Korea). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) including 10% fetal bovine serum, 2 mmol/L glutamine and 100 U/mL penicillin/streptomycin. The normal primary astrocytes were prepared from the neonatal rats, as described previously 58 , and cultured in Minimum Essential Media (MEM) with 10% FBS, 2 mmol/L glutamine and 100 U/mL penicillin/ streptomycin. plasmids, transfection and luciferase assay. The expression plasmids of survivn (pCA-flag-survivn) and cFLIP L (pCA-flag-cFLIP L ) were gift from Dr. Taeg K. Kwon (Keimyung University, Daegu, Korea). The point-mutant of cFLIP L at lysine 167/195 (cFLIP L -K167/195 R) was generated by a Quickchange TM Site-Directed Mutagenesis kit as previously described 59 . For luciferase assay, LN428 cells were transiently co-transfected with p2xNF-kB-Luc and pRSV-β-galactosidase into 1 × 10 6 cells per well in 6-well plates for 24 h using Lipofectamine reagent (Invitrogen). Cell were then treated with TNF (30 ng/ml) or TRAIL (50 ng/ml) for an additional 6 h, and the luciferase activities were measured using a luciferase assay kit (Promega, Madison, CA, USA) according to the manufacturer's instructions. Luciferase activity obtained were normalized to β-galactosidase activity of each sample. cell death assessment. Cell death was determined using Cell Titer-glo Luminescent Cell Viability Assay kit (Promega Co., Fitchburg, WI, USA), according to the manufacturer's instructions. Luminescent signals were measured by Tecan Infinite Plate reader (Tecan group Ltd., Männedorf, Switzerland), and Viability rates were calculated following the formula: viability rates = (1 − medicating/control) × 100%. Representative images were also taken by an inverted microscope. flow cytometry analysis. After LN428 cells were treated with pinoresinol, as described in the figure legends, the cells were harvested and examined for the mode of apoptotic and necrotic cell death by double staining with FITC-conjugated annexin V and propidium iodide (PI) in 10 mM HEPES buffer, pH 7.4, according to the manufacturer's instructions (BD FITC Annexin V kit). For cell cycle analysis, cells were suspended with ice-cold phosphate buffered saline (PBS) and fixed in 70% ethanol. Cells were washed with PBS and added 100 μg/ml PI solution including 50 μg/ml RNase in PBS for 30 min at room temperature. The cells were analyzed with a FACScan flow cytometer (BD Biosciences).
In vitro translation analysis. In vitro coupled transcription and translation analysis was performed with the 1-Step Human Coupled IVT Kit ± DNA (ThermoFisher Scientific) following the manufacturer's instructions. Briefly, the translation reaction was assembled with pre-incubation of HeLa cell lysates with 1 μg of circular DNA (pCFE-GFP) as a template. The reaction mixtures were incubated for 12 hours at 30 °C and were stopped with loading buffer for SDS-PAGE. The expression levels of EGFP proteins were examined by immunoblot analysis using anti-TurboGFP antibody. For in vitro translational analysis, EGFP mRNA was synthesized after digesting pcGlobin2-EGFP with XhoI, as described previously 60 . Synthetic EGFP mRNA was generated by using a mMES-SAGE mMACHINE T7 Transcription kit (ThermoFisher Scientific) following manufacturer's instructions. Each