Fig 2: Tubulin/microtubule regulation of DR5 mediated apoptosisTubulin and DR5 are found to form stable complexes which appears to promote DR5 protein degradation via undefined pathways. Pharmacological disruption of tubulin network using either depolymerizing (e.g., colchicine) or stabilizing (e.g. taxol) agent increases DR5 total and surface expression. In both cases, the resultant cells are sensitized to TRAIL induced apoptosis. Our data suggest distinct mechanisms: 1) Tubulin depolymerization appears to lower affinity to DR5 which results in release and stabilization of DR5 protein, and 2) Tubulin stabilization shows little or effect on DR5-tubulin complex formation, but it may act via inducing DR5 transcriptional expression [21]. In either case, the upregulated DR5 protein levels may be responsible for the increased surface expression and subsequent apoptosis in response to TRAIL treatment.

Fig 3: Silencing tubulin increases DR5 expression and TRAIL induced apoptosis(A) Cells were transfected with control siRNA (-) or siRNA against α-tubulin (+) for 48 hr, followed by immunoblot analysis. (B) Relative levels of cell surface DR5 was determined by flow cytometry. Cells were prepared as in (A) and stained with PE-conjugated monoclonal anti-DR5 antibody or isotype-matched control IgG (IgG2b). Data shown is relative to IgG2b-PE isotype controls. Values are means ± SD, **p<0.01, N=3. (C & D) Cells were transfected with siRNA as in (A), treated with TRAIL at 10 ng/ml for 24 h, followed by analyses for cell viability (C) and caspase activation (D).

Fig 4: Asiatic acid cause cis NPC-039 and cis NPC-BM cell apoptosis thought the intrinsic pathway and the extrinsic pathway. (a) Effect of Asiatic acid on mitochondrial membrane potential was analyzed by flow cytometric (b) The related mitochondrial membrane depolarized levels compared with control. (c) The expression levels of Fas, TNF-R1, DcR2, and DR5 were detected by Western blot. (d) The relative band density was normalized to GAPDH as a densitometer. * p < 0.05 versus control group.

Fig 5: Impact of TRAIL on A549 and dTHP‐1 sensitivities to ricin. (A) Ricin +TRAIL co‐treatment sensitizes A549 cells to ricin‐induced cell death (AUC of 5278 ±321 vs. 36,190 ±4027 for ricin‐only treated), but (B) TRAIL has no effect on ricin cytotoxicity in dTHP‐1 cells (AUC of 1924 ±223 vs. 2161 ±167 for ricin‐only treated). (C) TRAIL co‐treatment failed to influence ricin‐induced dTHP‐1 cell death even at much higher doses (12.5 ng/ml ricin). (D) Western blots analysis of A549 and dTHP‐1 cells demonstrate DR5/TRAILR2 expression in both cell types, the primary death receptor for TRAIL (Cell Signaling Technology Cat# 8074, RRID:AB_10950817). α‐tubulin served as the loading control (Cell Signaling Technology Cat# 5346, RRID:AB_1950376). Closed symbols, ricin‐only; open symbols, dual treatment, error bars represent the 95% CI. Green lines and dots and red lines and dots represent the mean and 95% CI of control‐ and ricin‐treated cells, respectively. Orange lines and dots represent the independent effect of TRAIL. Data presented are the mean of eight replicate wells with the 95% confidence interval. Graphical representation of the unnormalized data and results of pairwise statistical testing for treatment interactions are available in Figure S10. Refer to Figure S11 to observe the original films from which DR5 blot images were derived