Figure legends supplementary figures
Figure S1: Down-regulation of XIAP sensitizes HCT116 wt cells for TRAIL-induced apoptosis and overcome TRAIL resistance of Bax/Bak deficient HCT116 cells.
24 h after down-regulation of XIAP by siRNA, HCT116 wt and HCT116 Bax-/Bak- cells were treated with indicated concentrations of TRAIL and cultured for additional 24 h. Cells were harvested and induction of apoptosis was measured by Annexin V/PI staining. Cells positive for phosphatidylserine exposure and negative for PI staining represent early apoptotic cells; late apoptotic cells were characterized by detection of Annexin V and PI positivity.(A) TRAIL induced apoptosis in HCT116 wt cells in a dose dependent manner as evidenced by the detection of cells early apoptotic and detection late apoptotic cells (upper panel). As in the case of the DNA fragmentation analysis, the apoptotic rate was increased upon XIAP down-regulation (lower panel) (representative experiment). (B) In contrast to HCT116 wt cells, Annexin V positive cells were hardly detectable in the TRAIL-treated HCT116 Bax-/Bak- cells (upper panel), confirming their complete TRAIL resistance. Down-regulation of XIAP strongly sensitized the Bax/Bak deficient HCT116 cells for TRAIL-induced exposure of phosphatidylserine (lower panel) (representative experiment), showing that cells deficient for both Bax and Bak become susceptible to TRAIL-induced apoptosis when XIAP is inactivated. (C) Means +/- SD from 3 experiments are shown.
Figure S2: XIAP silencing overcomes TRAIL resistance of Bcl-2 and Bcl-xL over-expressing cells.
(A) To analyze the role of anti-apoptotic Bcl-2 family members on TRAIL resistance we generated HCT116 cell lines stably over-expressing Bcl-2 or Bcl-xL and corresponding HCT116 control cells by use of the respective pIRES vectors. Over-expression of Bcl-2 or Bcl-xL was confirmed by Western blot analysis.
(B) HCT116-control, HCT116-Bcl-2 and HCT116-Bcl-xL cells transfected with control or XIAP siRNA were treated with TRAIL and apoptosis was measured by flow cytometry. The percentage of hypodiploid, apoptotic cells is indicated between markers. TRAIL induced apoptosis in about 24 % of HCT116-control cells transfected with control siRNA. Induction of apoptosis was completely blocked by the over-expression of Bcl-2 or Bcl-xL, respectively, and less than 5 % of the cells showed a hypodiploid phenotype. Interestingly, down-regulation of XIAP not only strongly sensitized HCT116-control cells for TRAIL-induced apoptosis but also overcame resistance of Bcl-2 and Bcl-xL over-expressing cells. In detail, 45 % of the HCT116-control cells and around 38 % of both HCT116-Bcl-2 and HCT116-Bcl-xL cells exhibited an apoptotic phenotype. (C) Means +/- SD of the percentage of hypodiploid cells from three experiments.
Figure S3: Time course of phosphatidylserine exposure and PI positivity in Bax/Bak proficient HCT116 cells as compared to re sensitized Bax/Bak deficient XIAP knock-down HCT116 cells.
(A) HCT116 wt cells transfected with control siRNA and (B) HCT116 Bax-/Bak- cells transfected with XIAP siRNA were cultured in the presence of 50 ng/ml TRAIL for the indicated time. Induction of apoptosis was measured by Annexin V/PI staining as described in material and methods. Cells positive for phosphatidylserine exposure and negative for PI staining represent early apoptotic cells. Late apoptotic cells are characterized by detection of Annexin V and PI positivity. FACS analysis revealed that onset of TRAIL-induced apoptosis is comparable in both cell lines as determined by Annexin V-FITC positivity. However, TRAIL-treated HCT116 wt cells tend towards a late apoptotic phenotype, i.e. earlier onset of PI positivity. (C) Means +/- SD from 3 experiments are shown.
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