Figure 5.
Treatment with SMs results in the upregulation of cIAP2 by non-canonical activation of NF-κB. (a–c) cIAP2 protein levels increase after prolonged treatment with the SM. (a) HT1080 cells were incubated with 1 μM LBW242 or 100 nM Comp. A for the indicated time points. One hour before harvesting and lysing cells, fresh SM compounds were added (re-challenged) in the indicated lanes (LBW242 in lane and Comp. A in lane). (b) MDA-MB-231 cells were treated with 1 μM LBW242 or 100 nM of Comp. A for the indicated time points. (c) Me4405 and A2058 cells were treated for 16 h with 100 nM of Comp. A. (d) Cells were transfected with the indicated siRNA oligos, harvested and lysed 2 days after transfection. (e) cIAP2 mRNA levels increase upon cIAP1 depletion. BE cells were transfected with the indicated siRNA oligos and analysed after 48 h using quantitative RT-PCR. (f and g) SM treatment and siRNA-mediated depletion of cIAP1 and TRAF2 result in the activation of non-canonical NFκB signalling and induction of cIAP2 gene expression in BE cells. Cells stably expressing a luciferase reporter under the control of the cIAP2 promoter (cIAP2-Luc) were transfected with the indicated siRNA oligos and analysed for luciferase activity 48 h later. (h) SM treatment results in NIK-dependent activation of NF-κB. HT1080 cells stably expressing a NF-κB luciferase reporter were transfected with the indicated siRNA oligos and analysed for luciferase activity 48 h later. (i and j) SM- and TNFα-mediated induction of cIAP2 expression is NF-κB dependent. (i) HT1080 and HT1080IκB−SR cells, which express a non-degradable form of IκB, were transfected with the cIAP2-Luc reporter plasmid and left untreated or treated with the indicated combinations. (j) Inhibition of SM-mediated induction of cIAP2 results in elevated levels of NIK. HT1080 and HT1080IκB−SR cells were left untreated or treated with 1 μM LBW242 for 16 h