Fig. 5.

Depletion of XIAP re-sensitizes melanoma cells to IZI1551. a IZI1551-conditioned A375 melanoma cells were transiently transfected with an IκBα super repressor (IκBα-SR) or the empty vector (mock) and treated with IZI1551 (50 ng/ml). After 16 h apoptosis was determined using a CDDE (*p ≤ 0.05; n.s. = not significant) and b the status of IκBα, p-p65(Ser536), p65, and XIAP monitored by Western-blot analysis. GAPDH served as loading control. c Transcription was inhibited in parental and IZI1551-conditioned A375 cells by addition of Actinomycin D (ActD, 1 µM) for the indicated time points. Protein level of XIAP was monitored by Western-blot analysis. GAPDH served as loading control. d XIAP was depleted from parental and IZI1551-conditioned A375 and f Malme3M cells using RNAi for 72 h. Sixteen hours after treatment with IZI1551 (50 ng/ml) apoptosis was determined using a CDDE (***p ≤ 0.001) and e, g the status of XIAP, p-p65(Ser536), and p65 monitored by Western-blot analysis. β-actin served as loading control. h Parental and IZI-conditioned A375 melanoma cells were treated with IZ1551 (50 ng/ml) or increasing doses of the smac mimetic SM83 (0.1, 1, 10 µM) alone or in combination. After 16 h apoptosis was determined using a CDDE (**p ≤ 0.01) and i the status of XIAP monitored by Western-blot analysis. GAPDH served as loading control. j For five unstimulated mutBRAF melanoma cell lines the relative expression of XIAP was determined by semi-quantitative Western-blot analysis and k the apoptotic response to IZI1551 (50 ng/ml) determined after 16 h using a CDDE (***p ≤ 0.001). For CDDE the mean ± SD of three independently performed experiments is shown. WBs represent one out of three independently performed experiments