1. PHAPI, CAS, and Hsp70 Promote Apoptosome Formation by Preventing Apaf-1 Aggregation and Enhancing Nucleotide Exchange on Apaf-1 
Hyun-Eui Kim, Xuejun Jiang, Fenghe Du, Xiaodong Wang 
Molecular Cell 
Volume 30, Issue 2, Pages (April 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1 Cell-free Assay for Mediator of PHAPI
(A) Recombinant Apaf-1 was incubated with Apaf-1 null MEF cell extract (S100) or buffer A at 4°C for 2 hr and repurified using nickel beads. The repurified Apaf-1 from buffer A (left) or Apaf-1 null MEF S100 (right) was then incubated with cytochrome c, dATP, and purified recombinant procaspase-9 and procaspase-3 for caspase-3 activity measurement as described in the Experimental Procedures. The data represent the average fluorescence reading of two independent experiments, and the error bar indicates the standard deviation.
(B) Coomassie blue staining of purified proteins used in the assay. The production and purification of these proteins were described in the Experimental Procedures. The following proteins were used for SDS-PAGE and Coomassie blue staining: purified endogenous Apaf-1 from HeLa cell S100 (0.5 μg), purified recombinant procaspase-9 and procaspase-3 (0.5 μg each), purified bovine cytochrome c (1 μg), and purified recombinant PHAPI (0.5 μg).
(C) Assay for the PHAPI mediator. HeLa cell S100 was fractionated according to the diagram. Ninety percent ammonium sulfate solution was added to the Apaf-1-containing fraction from the Q-Sepharose column to 40% saturation, and the precipitated protein was collected by centrifugation. The pellet (P) was resuspended in buffer A (see the Experimental Procedures) and dialyzed against buffer A. Aliquots of it were then used to measure stimulated caspase-3 activation by PHAPI as indicated. All reaction mixture also contained dATP, purified endogenous Apaf-1, purified bovine cytochrome c, and recombinant procaspase-9 and procaspase-3. The assay was performed as in (A).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Purification and Characterization of PHAPI Mediator CAS
(A) Diagram of purification scheme for PHAPI mediator. See the Experimental Procedures and Results for details.
(B) Aliquots of 20 μl of fractions from the last heparin column were subjected to 10% of SDS-PAGE analysis followed by silver staining (Invitrogen). Black arrows point to recombinant Apaf-1. White arrows point to the proteins eluted at the same fraction as Apaf-1. These protein bands were cut out of the gel and subjected to mass spectrometric analysis.
(C) Titration of recombinant CAS for PHAPI-dependent caspase-3 activity. Recombinant human CAS was generated and purified as described in the Experimental Procedures. An aliquot of 1 μg of purified recombinant CAS was applied to 10% SDS-PAGE followed by Coomassie blue staining (right panel). In the left panel, increasing amounts of CAS were added to the reaction mixture that also contained the same proteins showed in Figure 1B and dATP. The caspase-3 activity was measured in the presence or absence of PHAPI as indicated.
(D and E) HeLa cells were transfected with two sets of siRNA against CAS at the indicated concentrations as described in the Experimental Procedures. The transfected cells were harvested 48 hr after transfection, and S100 was prepared as described in the Experimental Procedures. (D) Aliquots of 20 μg of S100 from each sample were analyzed by western blotting for CAS and actin levels. (E) Equal amounts of S100 from HeLa cells transfected with control siRNAi or SetI siRNA (100 nM) were applied to a HiTrap Q column, and Apaf-1-containing fractions were collected. Aliquots of 6 μl of Apaf-1-containing fraction were incubated with cytochrome c, dATP, procaspse-9, and procaspase-3. Caspase-3 activity was measured in the presence or absence of PHAPI and/or CAS as indicated. The data represent the average fluorescence reading of two independent experiments at 90 min at 30°C, and the error bar indicates the standard deviation. The experiment was repeated with setII siRNA against CAS and similar results were obtained (data not shown).
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4. Figure 3 In Vitro Reconstitution of Apoptosome-Regulatory Pathway
(A) Identification of Hsp70 as an Apaf-1-associated factor. Aliquots of 30 μl of indicated concentrations of purified endogenous Apaf-1 from HeLa cell extracts (lanes 1–3), recombinant Apaf-1 (lanes 4–6), and recombinant Hsp70 (lanes 7–9) were subjected to western blot analysis using antibodies against Apaf-1 and Hsp70.
(B) Titration of recombinant Hsp70 for PHAPI/CAS-dependent caspase-3 activity. Indicated concentrations of purified recombinant Hsp70 were added to the reaction mixture that also contained dATP and recombinant Apaf-1, procaspase-9, procaspase-3, cytochrome c, and PHAPI in the presence or absence of purified recombinant CAS. The caspase-3 activity was measured using a fluorogenic substrate as described in the Experimental Procedures.
(C) An aliquot of 1 μg of each purified recombinant protein was applied to 12% SDS-PAGE followed by Coomassie blue staining. rApaf-1, purified recombinant Apaf-1; Procasp-9, procaspase-9; Procasp-3, procaspase-3; Cyt.c, cytochrome c; Hsp70AAAA, Hsp70 mutant protein that has the last four amino acids changed to four alanines at the C terminus. All the recombinant proteins were generated and purified as described in the Experimental Procedures.
(D) Total reconstitution assay of CAS/PHAPI-dependent caspase-3 activity. The indicated proteins were added to the reaction mixture that contains recombinant Apaf-1, procaspase-9, procaspase-3, cytochrome c, and dATP. The caspase-3 activity was measured using flouorogenic substrate as described in the Experimental Procedures. The data represent the average fluorescence reading of two independent experiments at 90 min at 30°C, and the error bar indicates the standard deviation.
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5. Figure 4 CAS and PHAPI Enhance Nucleotide Exchange on Apaf-1
(A) TLC analysis of dATP loading on Apaf-1. The proteins indicated (lanes 6–20) were added to the reaction mixture that also contains purified recombinant Apaf-1, cytochrome c, radio-labeled dATP ([α-33P] dATP), and Hsp70. At indicated time points, the radioactive dATP was analyzed by a thin-layer chromatography (TLC) plate as described in the Experimental Procedures. In lanes 22–25, the protein indicated was omitted from the full mixture as in lane 21 and analyzed on the same TLC plate after 90 min of incubation. The nucleotide was visualized after exposing the TLC plate to an X-ray film.
(B) Quantification of nucleotide exchange by rapid filter binding assay. The reaction mixture containing purified recombinant Apaf-1, cytochrome c, and radio-labeled dATP was incubated with other proteins as indicated in the graph and applied to a rapid filter binding assay as described in the Experimental Procedures at the indicated time points. The filters were subjected to scintillation counting (cpm, counts per minute). The slope at the linear range (0–30 min) represents the nucleotide exchange rate. Nucleotide exchange rate increased 23-fold in the presence of Hsp70/PHAPI/CAS (47 cpm/s) compared to that of control (1091 cpm/s). The data represent the average radioactivity counting of two independent experiments, and the error bar indicates the standard deviation.
(C) Apoptosome formation and caspase-9 activation using recombinant proteins. The mixture of indicated recombinant proteins was incubated at 30°C for 1 hr before subjected to a Superose 6 gel filtration column. Apoptosome formation was visualized by western blotting analysis of the column fractions using antibodies against Apaf-1 and caspase-9. The column was equilibrated with a set of known molecular size markers, and the positions at which the 670 and 158 kDa markers were eluted were indicated.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Inducible Knockdown of CAS Blocks UV Irradiation-Induced Apoptosis
(A) CAS expression is induced by UV irradiation. Aliquots of 20 μg of S100 from HT-29 cells treated with the indicated dose of UV irradiation were applied to SDS-PAGE and analyzed by western blotting using antibodies against CAS and Apaf-1.
(B and C) HT-29 cells that stably express tetracycline-inducible CAS shRNA were generated as described in the Experimental Procedures. Cells were pretreated with or without tetracycline for 48 hr. Cells were then treated with 100 mJ/cm2 UV for an additional 24 hr. (B) Cell survival was measured by methylene blue staining as described in the Experimental Procedures (left panel). The levels of CAS and actin after 48 hr of tetracycline treatment were analyzed by western blotting using antibodies against CAS and actin (right panel). (C) S100 was prepared from cells treated with or without tetracycline and UV as in (B) and subjected to a Superose 6 gel filtration chromatography. The column fractions were probed with an anti-Apaf-1 antibody. The column was equilibrated with a set of known molecular size markers, and the positions at which the 670 and 158 kDa markers were eluted were indicated.
(D) CAS-shRNA stable cells were transfected with either vector (V) or scrambled CAS expression plasmid with the shRNA targeting site silently mutated (S). Twenty-four hours later, tetracycline was added to the culture media as indicated. After an additional 48 hr, cells were treated with UV (100 mJ/cm2), and cell survival rate was measured 24 hr later (upper panel). The expression of CAS (endogenous and scrambled) as well as actin was determined by western blotting (lower panel). The data represent the average percentage of cell survival of two independent experiments, and the error bar indicates the standard deviation (B and D).
Molecular Cell  , DOI: ( /j.molcel )
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