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Leu and George

Hepatic IGFBP1 is a Prosurvival Factor that Binds to BAK,

Protects the Liver from Apoptosis, and

Antagonizes the Proapoptotic Actions of p53 at Mitochondria

J. I-Ju Leu and Donna L. George

Department of Genetics, University of Pennsylvania School of Medicine, 415 Curie Blvd., Philadelphia, Pennsylvania, 19104

Supplemental Material

Figure legends

Figure S1. IGFBP1 is a BAK binding protein. (A) In vitro translated IGFBP1 was mixed with in vitro translated, 35S-labeled BAD, BAX, BAK, or BCL-xL. The upper panel confirms similar expression levels of these BCL2 family proteins (input), while the bottom panel shows interaction of IGFBP1 only with the BAK protein. (B) (Top panel) In vitro translated 35S -labeled wild type (WT) BAK, or the 35S-labeled BH-domain BAK mutants indicated, show equal expression. (Middle panel) The labeled BAK proteins were individually incubated with unlabeled in vitro translated IGFBP1, and the products subjected to IP with anti-IGFBP1 antibody before SDS-PAGE. (Bottom panel)In vitro translated 35S-labeled wild type (WT) BCL-xL was mixed individually with the unlabelled BAK protein forms indicated. The products were subjected to IP with anti-BAK antibody. (C) Total (T), cytosolic (C), and mitochondrial (M) fractions were isolated from HepG2 cells before and after treatment with 3.5 μM cisplatin (L-Cis) or 10 μM Nutlin-3. Samples were examined by immunoblotting for the presence of the proteins indicated.

Figure S2. The level of IGFBP1 expression varies indirectly with apoptosis induction. (A) Western blot analysis of protein expression following treatment of the HepG2 cells with the indicated amount of cisplatin, doxorubicin, or Nutlin-3. The appearance of cleaved products of PARP indicates apoptosis, which also correlates with the morphological appearance of the cells (data not shown). (B) This is the same data as shown in Fig. 2B, but it also includes a shorter exposure of the BAK immonoblot. (C) This is the same data as shown in Fig. 2D, but it also includes a longer exposure of immunoblots showing the BAK/p53 interaction as well as the interaction between IGFBP1 and BAK. (D) IP-western blot analysis reveals that p53 co-IPs with BAK following H-Cis (50 μM), but not L-Cis (3.5 μM), treatment of HepG2 cells. In contrast, cisplatin did not alter the relative abundance of the BCL-xL/BAK complex. The left panel provides input analysis of the protein expression levels, while the right panel shows the results of the immunoprecipitation analyses. (E) IP-western blot analysis confirms the presence of a p53/BAK complex in doxorubicin (Dox, 4 μM)-treated HepG2. The left panel provides input analysis of the protein expression levels, while the right panel shows the results of the immunoprecipitation analyses. (F) The same data as shown in Fig. 3C, showing longer exposure of BAK/p53 co-IP.

Figure S3. Response of primary cultures of MEFs to cisplatin (50 μM, 15 h). (A) Representative field showing senescence-associated -galactosidase (SAGal) staining of early passage (pass 4) BAK+/+ or BAK-/- MEFs, either before or after cisplatin treatment (50 μM, 15 h). The blue staining indicates positive SAGal staining. Scale bars: 50 μm. (B) Quantification of SAGal-positive cells, based on the average number observed in eight random 20X fields + s.d.

Figure S4. IGFBP1 expression antagonizes p53/BAK interaction and p53-mediated BAK oligomerization. (A) IP-Western blot reveals a p53/BAK complex in WCE prepared from MCF7 cells treated with doxorubicin (Dox, 2 μM). In this assay, apoptosis was assessed by the relative loss/cleavage of the full-length form PARP. (B) Western blots of the indicated proteins in WCE or mitochondrial fractions (Mito) prepared from untreated- or Dox (2 μM)-treated MCF7 and MCF7-IGFBP1 cells. Note the relative loss/cleavage of the full-length form PARP in the Dox-treated MCF7, compared to the MCF7-IGFBP1 cells. (C) The same data as that in Fig. 4F, but including a longer exposure of p53/BAK co-IP. (D) The same data as in Fig. 4G, but including a lighter exposure of p53-mediated BAK oligomerization. Note mitochondria from IGFBP1-expressing HepG2 cells display a dose-dependent resistance to the p53-mediated BAK oligomerization. (E) Western blots of the indicated proteins in Mito fractions prepared from HepG2 untreated, or treated with 10 μM Nutlin-3, as well as parental U2OS and U-BP1 cells. (F) 20 μg of Mito prepared from U2OS or U-BP1 cells were incubated with 50 pmol of purified p53-recombinant protein. The reactions were crosslinked with 5 mM BMH (the uncleavable protein crosslinker 1,6-bismaleimidohexane) followed by western analysis using an anti-BAK antibody (a conformation-specific antibody directed to the N-terminus). (G) Quantification of TUNEL-positive cells in control wild-type (WT) liver, wt liver 24 h after -amanitin-treatment, control IGFBP1-/-(BP1-/-) liver, BP1-/- liver 24 h after -amanitin-treatment, control BAK-/- liver, BAK-/- liver 24 h after -amanitin-treatment, and p53-/- liver 24 h after -amanitin-treatment. Average of six random 20X fields + s.d. The results shown are representative of the analysis of at least three mice per genotype.