Figure 1.
Reduced Activation of Caspase-3 in Infected Cells
(A) HeLa cells were infected (+) for 16 h or not (−) and treated for an additional 8 h with TNFα and CHX. Lysates of these cells were separated and probed with an antibody specifically recognizing the active p19/17 fragment of caspase-3 by immunoblot analysis.
(B) Blots from the same lysates used in (A) were probed for caspase-8 (casp-8 p18) and caspase-3 (casp-3 p19/17). The processing of BID as a specific substrate for caspase-8 is also shown.
(C) HeLa cells were infected and treated with TNF/CHX as mentioned before. Caspase-8 p60 (Casp-8) and −3 p32 (Casp-3) was detected by immunoblot analysis of the lysates. β-Actin staining was included as equal loading control.
(D) Active caspase-8 and caspase-3 were precipitated from the same lysates as in (A) using Biotin-VAD-fmk, and precipitated proteins were detected with antibodies for caspase-8 and caspase-3 as indicated.
(E) Active caspase-8 was monitored in living HeLa cells infected with Ctr and treated with TNFα/CHX (TNF) as described in Protocol S1 (Detection of active caspases). Shown is the mean ± SD of three experiments.
(F) Caspase-3 p19 and p17 fragments were detected by immunoblotting in lysates of cells infected for the indicated times with Ctr and treated for 5 or 10 h with TNFα/CHX.
(G) Cells infected for the indicated time points with Ctr were treated with TNFα/CHX for 10 h, and the full-size caspase-8 (p60) and the cleavage products (p41/43; p18) were analyzed by immunoblotting.
Figure 2.
Infection-Induced Up-regulation of cIAP-2
(A) mRNA levels of different IAPs were monitored by qRT-PCR in HeLa cells infected (+) or not (−) with Ctr. The amount of cIAP-2 mRNA increases strongly in infected cells.
(B) The level of IAP expression was determined by immunoblot analysis in noninfected (−) and infected (+) cells. Note that cIAP-2 and survivin are strongly up-regulated in infected cells.
(C) Time course of cIAP-2 and survivin up-regulation was tested in infected cells by immunoblot analysis. Cells were infected and lysed at different time points postinfection as indicated.
Figure 3.
IAPs Required for Infection-Induced Apoptosis Resistance
(A) HeLa cells were transfected with control siRNA (siLam) or siIAP-2 and infected (+ Ctr) or not (− Ctr) with Ctr. Then, apoptosis was induced by treatment with TNF/CHX, and the apoptotic response was measured by phase contrast (PC) and TUNEL analysis. Arrows point to Ctr inclusions.
(B) Infection-induced up-regulation of cIAP-2 prevents the processing of the caspase-3 p19 to the p17 fragment. HeLa cells were transfected with siRNAs, infected, and treated with TNF/CHX as indicated. cIAP-2 and the p19 and p17 fragment of caspase-3 were monitored by immunoblotting on the same membrane. The level of cIAP-2 in the siIAP-2 and TNF/CHX-treated infected sample roughly equals that of the uninfected siLuc and TNF/CHX-treated samples. Note that the proportion of the p19 and p17 fragments in these samples is similar but is clearly shifted in the other TNF/CHX-treated samples. The proportion of caspase-3 p19 and p17 fragment was calculated by densitometric analysis and outlined below the immunoblot (in %).
(C) The apoptotic response was determined in cells transfected with the indicated siRNAs and either infected (+) or not (−) by TUNEL assay. Any one of the siRNAs directed against cIAP-1, cIAP-2, and XIAP, but not the control and the survivin siRNA, strongly sensitizes infected cells for TNF-induced apoptosis. Shown is the mean ± SD of three experiments.
Figure 4.
IAPs Are Organized in Heteromeric Complexes
(A) Endogenous cIAP-2 and XIAP were immunoprecipitated (IP) from control and apoptotic cells as indicated. The level of endogenous cIAP-2 and XIAP was determined in precipitates by immunoblotting.
(B) Endogenous IAPs were precipitated from control and TNF/CHX-treated cells and the coprecipitation of active caspase-3 was monitored by immunoblotting.
(C) Endogenous cIAP-2 was immunoprecipitated from control and infected cells treated or not with TNF/CHX as indicated. The level of endogenous cIAP-2 and XIAP was determined in precipitates (IP) and in lysates.
(D) The IAP complexes were isolated by gel filtration as mentioned in Materials and Methods. The proteins from different fractions were TCA-precipitated and resolved in SDS-PAGE, and the presence of IAPs was checked by immunoblot analysis as indicated. Note that high amounts of cIAP-1, XIAP, and cIAP-2 were present together in a protein complex of around 400 kDa. Shown are the fraction numbers (fractions) and the approximate size of proteins and complexes in the respective fractions in kDa.
(E) The cytosol from HeLa cells induced to apoptosis with TNF/CHX was isolated by subcellular fractionation, and gel filtration was performed as mentioned in Materials and Methods. The proteins were separated by SDS-PAGE, and the immunoblot analyses were performed as before.
(F) Gel filtration experiments were performed in XIAP-silenced cells and the presence of cIAP-1 and cIAP-2 in various fractions was tested by immunoblot analysis. Shown in (C), (D), and (E) are the data from one representative experiment.
Figure 5.
IAPs Cross-Regulate Each Other
siRNAs directed against (A) luciferase or XIAP, (B) cIAP-1, (C) cIAP-2, or (D) survivin were transfected in HeLa cells, and the protein levels of all IAPs were determined in infected (+) or uninfected (−) samples by immunoblot analysis as indicated.