Fig 2: Proposed schematic of the roles of NIK and SMG1 in sensitive versus resistant cells in response to SMC and TNFα treatment. In SMC sensitive cells (left side), removal of the cIAPs by SMC treatment prevents ubiquitination of RIP1, allowing RIP1 to bind and activate the death complex II that includes caspase-8 and FADD (A). Degradation of SMG1 allows for the activation of JNK in response to TNFα, leading to proteasomal downregulation of c-FLIP possibly through the ITCH-mediated pathway (B). As c-FLIP is downregulated, caspase-8 activation is able to proceed and apoptosis ensues. In SMC-resistant cells (right side), the level of SMG1 persists in spite of SMC and TNFα treatment, blunting the activation of JNK (C) and thereby preventing proteasomal degradation of c-FLIP. In addition, depletion of cIAPs by SMC treatment allows NIK to accumulate, resulting in activation of both the classical and alternative NF-κB pathways (D), which may elevate transcriptional levels of c-FLIP. SMG1 and NIK functions appear to converge by sustaining the protein expression level of c-FLIP, thereby preventing caspase-8 activation and repressing SMC-mediated apoptosis. Silencing of both SMG1 and NIK converts SMC-resistant cells to become sensitive to SMC treatment in response to TNFα

Fig 3: cIAP1, cIAP2 and XIAP cooperatively protect against TNFα-induced cell death in cancer cells depleted of SMG1 and NIK. (a) H226, (b) H460 and (c) H661 cells were transfected with siRNA targeting cIAP1, cIAP2, XIAP, SMG1, NIK or non-targeting siRNA as a control. At 48 h post-transfection, cells were treated with a vehicle (red bars), or with 100 ng/ml TNFα (blue bars) for an additional 48 h. (d) H226 cells were transfected with siRNA targeting SMG1, NIK or non-targeting siRNA as a control for 24 h, and then treated with combinations of 100 nM SM-164 and 100 ng/ml TNFα for an additional 48 h. Cell viability was determined by Alamar Blue. Numbers represent the average ±S.E.M. of at least three assays. *P<0.01 significantly different from non-targeting siRNA and vehicle, as determined by ANOVA with post hoc Tukey's HSD. (e) H226, H460 and H661 cells were transfected with siRNA targeting cIAP1, cIAP2 and XIAP. At 48 h post-transfection, cells were harvested and protein expression levels of cIAP1, cIAP2 and XIAP analyzed by western immunoblotting. β-Actin was used as a loading control

Fig 4: Depletion of SMG1 and NIK allows cancer cells to undergo apoptosis in response to SMC and TNFα treatment. (a) H226 (b) H460 and (c) H661 cells were transfected with siRNA targeting SMG1, NIK or non-targeting siRNA as a control. At 24 h after siRNA-mediated silencing, cells were exposed to combinations of 100 nM SMC-AEG40730 and 100 ng/ml TNFα. At 24 h after drug treatment, cells were stained with annexin V and propidium iodide to measure the rate of apoptosis by flow cytometry. Plotted numbers represent the average ±S.E.M. of three independent assays. *P<0.001 significantly different from vehicle for the respective siRNA group treated with vehicle, as determined by ANOVA with post hoc Tukey's HSD. (d) Representative dot plots of H460 cells treated as in (b). The downregulation of SMG1 and NIK promoted SMC-mediated TNFα-induced apoptosis in cancer cells

Fig 5: Depletion of SMG1 and NIK promotes SMC-mediated TNFα-induced caspase activation. (a) H226, H460 and H661 cells were transfected with siRNA targeting SMG1 (red bars), NIK (blue bars), both (green bars) or non-targeting (NT) siRNA (black bars) as a control. At 24 h after siRNA-mediated silencing, cells were exposed to combinations of 100 nM SMC-AEG40730 and 100 ng/ml TNFα for an additional 48 h. Cell viability was determined by Alamar Blue. Numbers represent the average ±S.E.M. of at least three assays. *P<0.01 significantly different from non-targeting siRNA and vehicle, as determined by ANOVA with post hoc Tukey's HSD. (b) H460 cells were transfected with siRNA targeting SMG1, NIK, or non-targeting siRNA as a control. At 24 h after siRNA-mediated silencing, cells were exposed to SMC for 1 h before the addition of 100 ng/ml TNFα. At the indicated times of TNFα treatment, cells were harvested and subjected to western immunoblotting with antibodies recognizing caspase-8, cleaved caspase-3, SMG1, NIK, cIAP1, cIAP2 and XIAP. β-Actin was used as a loading control. Processing of caspase-8 and -3 were detected in SMG1- and NIK-depleted cells following combined SMC and TNFα treatment. (c) H226 cells were transfected with siRNA targeting SMG1 and NIK, in combination with siRNA targeting caspase-8, -9 or RIP1 for 48 h, and then exposed to SMC and TNFα for an additional 48 h before cell viability was determined by Alamar Blue. Numbers represent the average ±S.E.M. of at least three assays. *P<0.001 significantly different from non-targeting siRNA and vehicle, as determined by ANOVA with post hoc Tukey's HSD. (d) H226 cells were treated with siRNA as in (c) for 48 h, and isolated proteins were probed by western immunoblotting for the presence of caspase-8, -9 and RIP1. The silencing of caspase-8 or RIP1 prevented SMC-mediated TNFα-induced cell death in dually NIK- and SMG1-depleted cells