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1.
Figure 2

Figure 2. Anti-tumorigenic effects of NuBCP-9s. From: A Short Nur77-derived Peptide Converts Bcl-2 from a Protector to a Killer.

(A) Effect of NuBCP-9s (10 µM) on clonogenic survival of MEFs and Bcl-2−/−MEF.
(B) Effect of NuBCP-9 (20 µM) on clonogenic survival of MEFs and Bax−/−Bak−/−MEFs.
(C) Effect of D-NuBCP-9 (15 µM) on clonogenic survival of MEFs and Bax−/−Bak−/−MEFs.
Cells were exposed to the indicated concentration of NuBCP-9-pen, D-NuBCP-9-pen, or NuBCP-9/AA-pen (Control). The data (A–C) are means ± SD from triplicates.
(D) Inhibition of tumor growth by NuBCPs. MDA-MB435 tumors grown in SCID mice (n=5) were injected with the indicated peptide, and tumor volumes were measured.
(E) Induction of apoptosis of tumor by NuBCPs. Tumor tissues from animals treated with the indicated peptide were stained by TUNEL for apoptosis induction. Scale bar=100 µM.
(F) Correlation of apoptosis induction and Bcl-2 conformational change in vivo. Apoptosis in the tumor tissues from animals treated for two days with peptide was determined by TUNEL staining. Conformational change in Bcl-2 was detected in the same tissues by immunostaining with anti-Bcl-2/BH3 antibody. Nuclei were visualized by DAPI staining. Scale bar=100 µM.
(G) Representative ultrasound images of established tumors injected with D-NuBCP-9. MDA-MB435 tumors (about 0.6 cm2 in size) grown in SCID mice were injected with PBS or D-NuBCP-9-pen, and tumors were monitored by ultrasound technology (VisualSonics Inc., Toronto). Scale bar = 125 mM.

Siva Kumar Kolluri, et al. Cancer Cell. ;14(4):285-298.
2.
Figure 5

Figure 5. NuBCP-9 and its enantiomer bind to the loop of Bcl-2. From: A Short Nur77-derived Peptide Converts Bcl-2 from a Protector to a Killer.

(A) Binding of Bcl-2/1-90 with Nur77 by Co-IP. Bcl-2 mutants used are shown the left panel. The indicated Nur77 mutant expression vector was cotransfected with Bcl-2/1-90 tagged with Myc epitope and their interaction was analyzed by Co-IP using anti-Myc antibody. Specific and nonspecific (NS) bands are indicated. Input represents 5% of cell lysates used in Co-IP assays. Data from one of three experiments is shown.
(B) FP assays. Bcl-2/1-90 were incubated with the indicated FITC-NuBCP-r8 (20 nM) in triplicate in PBS, pH 7.6 at 25°C and FP determined when the signal stabilized within 20 min. Competition experiments (lower panel) were carried out by incubating 200 nM GST-Bcl-2/1-90 protein with 20 nM FITC-NuBCP-r8 in the presence or absence of unlabeled NuBCPs in triplicate and FP determined within 20 min upon stabilization of the signal. Data are presented as mean ± SD.
(C) CD spectra for the binding of NuBCP-9s (30 µM) to GST-Bcl-2/1-90 (2 µM) in PBS, pH 7.6, 25°C. Data from one of the three experiments are shown.
(D) Stoichiometry of binding of NuBCP-9 to GST-Bcl-2/1-90. GST-Bcl-2 was titrated with NuBCP-9s in PBS, pH 7.6, 25°C at 219 nm where absorption from the free peptide is <0.3% of protein. Stoichiometry was determined as described (Jones et al., 2002). Data are presented as mean ± SD.
(E,F) Co-IP assay. HEK293T cells transfected with GFP-Nur77/DC3 and Myc-tagged Bcl-2/29-90 were exposed to NuBCP-9 or D-NuBCP-9 and their interaction analyzed by Co-IP. Input represents 5% of cell lysates used in Co-IP assays. Data from one of the three experiments are shown.
(G) Mutations or insertion in the loop of Bcl-2 affects loop binding to Nur77. Bcl-2 loop mutants were tagged with Myc epitope, and they were transfected into HEK293T cells with GFP-Nur77/DC3. Cell lysates were prepared and analyzed for interaction of Bcl-2 loop mutants with Nur77/DC3 by Co-IP. Input represents 5% of lysates used for Co-IP assays. Data from one of the three experiments are shown.
(H) FP assays. Bcl-2/29-90 was incubated with the indicated FITC-NuBCP-r8 (20 nM) in triplicate in PBS, pH 7.6 at 25°C and FP determined when the signal stabilized within 20 min. Competition experiments (lower panel) were carried out by incubating 200 nM GST-Bcl-2/29-90 protein with 20 nM FITC-NuBCP-r8 in the presence or absence of unlabeled NuBCPs in triplicate and FP determined within 20 min upon stabilization of the signal. Data are presented as mean ± SD.
(I) CD spectra for the binding of NuBCPs (30 µM) to GST-Bcl-2/29-90 (2 µM). Data from one of the three experiments are shown.

Siva Kumar Kolluri, et al. Cancer Cell. ;14(4):285-298.
3.
Figure 7

Figure 7. NuBCPs disrupt Bcl-2’s intra-molecular interaction and binding with tBid. From: A Short Nur77-derived Peptide Converts Bcl-2 from a Protector to a Killer.

(A) Intra-molecular interaction in Bcl-2. GFP-BH4 or Bcl-2/ΔBH4 was transfected alone or together into HEK293T cells. Their interaction was analyzed by Co-IP.
(B,C) Nur77 and NuBCP-9s disrupt Bcl-2 intra-molecular interaction. The indicated expression vectors were transfected into HEK293T cells. The effect of Nur77/ΔDBD and Nur77/DC3 (B) or NuBCP-9s (C) on interaction between BH4 domain and Bcl-2 was analyzed by Co-IP.
In A–C, input represent 5% of cell lysates used in the Co-IP assays, and representative data from one out of four independent experiments are shown.
(D) Removal of BH4 domain exposes BH3 domain. HEK293T cells transfected with either Bcl-2 or Bcl-2/ΔBH4 were stained with either anti-Bcl-2/BH3 or polyclonal anti-Bcl- 2 antibody. Scale, 10 µM.
(E) NuBCP-9 inhibits tBid/Bcl-2 interaction in liposomes. The membrane permeabilization induced by tBid/Bcl-2 interaction was monitored by the release of 0.5-kDa CB dyes from the liposomes after 3 hr incubation at 37 °C. The release resulted in the quenching of CB fluorescence by the anti-CB antibody located outside of the liposomes. The extent of the release was determined by the value of ΔFProtein/ΔFTriton as described in Supplemental Data. The effect of NuBCP-9 or NuBCP-9/AA peptide on the release was determined by including the corresponding peptide at indicated concentrations to the incubation. Data shown are means of 2 to 4 independent experiments with SD indicated by error bars.
(F) NuBCP-9-induced Bcl-2 conversion does not activate Bax pore activity in liposomes. The extent of the 0.5-kDa CB dye release by Bax and/or Bcl-2 in the absence or presence of NuBCP-9 peptide was monitored as above. Data shown are means of 2 to 5 independent experiments with SD (error bars).

Siva Kumar Kolluri, et al. Cancer Cell. ;14(4):285-298.
4.
Figure 3

Figure 3. NuBCPs interact with Bcl-2 and target mitochondria. From: A Short Nur77-derived Peptide Converts Bcl-2 from a Protector to a Killer.

(A) HEK293T cells transfected with GFP-Nur77/489-497 or GFP-Nur77/478-504 (2 µg) together with or without Bcl-2 expression plasmid (0.8 µg) were analyzed by co-immunoprecipitation (Co-IP) using anti-Bcl-2 antibody, followed by Western blotting (WB) using either anti-GFP or anti-Bcl-2 antibody (Lin et al., 2004). Cells were transfected in six-well plates at 80% confluency.
(B) HEK293T cells transfected with GFP-Nur77/ΔDBD and Bcl-2 were exposed to NuBCP-9 or D-NuBCP-9 and analyzed by Co-IP.
(C) Interaction of NuBCP with anti-apoptotic Bcl-2 family members, Bcl-2, Bcl-B and Bfl-1. HEK293T cells in six-well plates transfected with GFP-Nur77/478-504 (2 µg) together with or without the indicated Myc-tagged Bcl-2 family member expression plasmids (0.8 µg) were analyzed by Co-IP, followed by WB.
Input in panels A, B, and C represents 5% of cell lysates used in the Co-IP assays. Data from one of the three experiments are shown.
(D) FP assay of binding of NuBCPs to Bcl-2. 200 nM GST-Bcl-2, GST-Bcl-XL, or GST protein was incubated with the indicated FITC-NuBCP-r8 (20 nM) in triplicate in PBS, pH 7.6 at 25°C and FP determined when the signal stabilized within 20 min. Data are presented as mean ± SD.
(E) FP competition assay. 200 nM GST-Bcl-2 protein was incubated with the indicated FITC-NuBCP-r8 (20 nM) in the presence or absence of unlabeled NuBCPs in triplicate in PBS, pH 7.6 at 25°C and FP determined when the signal stabilized within 20 min. Data are presented as mean ± SD.
(F) NuBCPs target mitochondria. RFP-Mito (50 ng) was transfected into H460 cells on glass cover slips together with GFP-Nur77/489-497 or GFP-Nur77/478-504 (50 ng) for 12 hr, or RFP-Mito was transfected into H460 cells for 12hr and treated with 6 µM FITC-D-NuBCP-9-r8 for 2 hr, and subcellular localization of the fluorophores analyzed by confocal microscopy. Approximately 10–15% GFP-fusion-transfected or FITC-D-NuBCP-9-treated cells displayed the localization shown. Scale, 4 µM.

Siva Kumar Kolluri, et al. Cancer Cell. ;14(4):285-298.
5.
Figure 4

Figure 4. NuBCPs induce Bcl-2 conformational change. From: A Short Nur77-derived Peptide Converts Bcl-2 from a Protector to a Killer.

(A) H460 cells were exposed to the indicated peptide (20 µM) for 12 hr, and endogenous Bcl-2 was immunoprecipitated by anti-Bcl-2/BH3 antibody, followed by WB using a polyclonal anti-Bcl-2 antibody against the whole protein. Input represents 10% of lysates used for IP.
(B) Flow cytometry analysis of endogenous Bcl-2 immunofluorescence. H460 cells were treated as in (A) and immunostained with anti-Bcl-2/BH3 antibody (Abgent) and SRPD-conjugated secondary antibody (Southern Biotech). Bcl-2 fluorescence from peptide-treated cells (green histogram) was compared to that from the non-treated cells (purple histogram).
(C) CD spectra for the binding of NuBCP-9s (30 µM) to GST-Bcl-2 (2 µM) in PBS, pH 7.6, 25°C. Data from one of the three experiments are shown.
(D) Stoichiometry of binding of NuBCP-9 to GST-Bcl-2. GST-Bcl-2 was titrated with NuBCP-9s in PBS, pH 7.6, 25°C. CD was taken at 219 nm where absorption from the free peptide is <0.3% of protein. Stoichiometry was determined as described (Jones et al., 2002). Data are presented as mean ± SD.
(E) Summary of binding of NuBCP-9 and its enantiomer to Bcl-2 and mutants. Kd ± SD values for FITC-NuBCP-9-r8s binding GST-Bcl-2, GST-Bcl-2/1-90 or GST-Bcl-2/29-90 were calculated from single-site FPA binding curves shown in Fig. 3D, Fig. 5B and 5H using Prism software. EC50 values for unlabeled NuBCP-9s binding the same Bcl-2 constructs were derived from single-site FPA competition curves shown in Figure 3E, Figure 5B and 5H using Prism software. χ2 values indicate excellent agreement with a single-site binding model. Ki values for the unlabeled NuBCP-9s were calculated from using Kd determined for the FITC-labeled peptides. Kd values were determined from CD binding curves (Figure 4D and Figure 5D) using nonlinear regression analysis for a one-site-binding model. Stoichiometry ± SD was determined as described (Jones et al., 2002).

Siva Kumar Kolluri, et al. Cancer Cell. ;14(4):285-298.
6.
Figure 1

Figure 1. Apoptosis induction by NuBCP. From: A Short Nur77-derived Peptide Converts Bcl-2 from a Protector to a Killer.

(A) Location of NuBCP in Nur77, Nur77 mutants and peptide sequences used in this study. Single letter code for amino acid (aa) with upper case for L-aa and lower case for D-aa. X, N-aminocaproic acid; CX, covalent linkage between cysteine thiol and acetyl group. Mutated aa is bolded. Peptides conjugated with polyarginine (r8) were used in all studies unless indicated.
(B) NuBCP-9 induces apoptosis in ZR-75-1 breast cancer but not in normal primary mammary epithelial cells. Cells were exposed to NuBCP-9 for 24 h and apoptosis determined by Annexin V staining.
(C) Apoptotic effect of NuBCP-9 is retained with its enantiomer. H460 cells were exposed to the indicated peptide for 24 h and apoptosis determined by Annexin V staining.
(D) Bcl-2 dependent apoptosis induction by NuBCP-9s in Jurkat cells. Bcl-2 expression in Jurkat cells transfected with neo control vector (Jurkat/Neo) or Bcl-2 expression vector (Jurkat/Bcl-2) was determined by immunoblotting (left panel). Cells were exposed to NuBCP-9s (10 µM) in media containing 5% FBS for 36 h, and apoptosis determined by DAPI staining.
(E) Knockout of Bcl-2 enhances the apoptotic effect of STS and Bad BH3 peptide. Bcl-2 expression in MEFs and Bcl-2−/−MEFs was determined by immunoblotting (left panel). Apoptosis of cells exposed to STS (0.1 µM) or Bad BH3 peptide (10 µM) for 36 h was determined by DAPI staining.
(F) Dose dependent apoptosis induction by NuBCP-9s in MEFs and Bcl-2−/−MEFs. Cells (10,000 cells/well) were seeded in medium containing 5% FBS and then exposed to the indicated concentration of NuBCP-9 peptides conjugated with penetratin peptide (NuBCP-9-pen) (Supplementary Figure 2) for 36 h. Apoptosis was determined by DAPI staining.
(G) Time-course analysis of apoptosis induction by NuBCP-9s in MEFs and Bcl-2−/− MEFs. Cells (15,000 cells/well) seeded in medium containing 5% FBS were exposed to the indicated NuBCP-9-pen (10 µM) for the indicated period of time. Apoptosis was determined by DAPI staining.
(H) Bax or Bak is required for apoptotic effect of NuBCPs. The indicated MEFs were exposed to peptide (15 µM) for 36 h, and apoptosis was determined by Annexin V staining.
In panels B–H, the bars represent means ± SD from 3–4 experiments

Siva Kumar Kolluri, et al. Cancer Cell. ;14(4):285-298.
7.
Figure 8

Figure 8. Induction of apoptosis by BH3 domain of Bcl-2. From: A Short Nur77-derived Peptide Converts Bcl-2 from a Protector to a Killer.

(A) BH3 peptide from Bcl-2 and its mutant. For cell-based assays, the peptides were fused with cell-penetrating peptide from Ant. For liposome-based study, the peptides only contained the BH3 residues.
(B) Dominant-negative effect of Bcl-2/BH3-L/A,D/A. H460 cells in six-well plates were transfected with GFP (1 µg) and empty control vector (2 µg) or GFP (1 µg) and Bcl-2/L97A/D102A (2 µg) for 24 hr prior to exposure to NuBCP-9 (10 µM) for another 16 hr. Apoptosis of transfected cells was determined by Annexin V staining. Expression of Bcl-2/L97A/D102A was confirmed by WB (not shown). Error bars represent mean ± SD.
(C) Bcl-2 BH3 peptide neutralizes the inhibitory effect of Bcl-XL on tBid-induced Bax membrane permeabilizing activity in liposomes. The tBid-induced Bax activity was monitored by the release of 10-kDa CB-dextrans from the liposomes after 3 hr incubation at 37°C. The effect of Bcl-XL and/or Bcl-2 BH3 or its mutant peptide on the tBid/Bax-mediated CB-dextran release was determined by including the corresponding protein and/or peptide to the incubation at indicated concentrations. Data shown are means of 2 to 3 independent experiments with SD (error bars).
(D) FP assay of binding of Bcl-2 BH3 peptide to Bcl-XL. The indicated concentration of GST-Bcl-XL or GST protein was incubated with F5M-Bcl-2 BH3 or F5M-Bcl-2 BH3-L/A,D/A (20 nM) in duplicate in PBS, pH 7.4 at 25°C and FP determined.
(E) Bcl-2 and NuBCP-9 reverses the inhibitory effect of Bcl-XL on Bax-dependent membrane permeabilization in liposomes. The effect of Bcl-XL (10 nM) in the presence of Bcl-2 (200 nM) and/or NuBCP-9 (10 µM) on the tBid (5 nM) /Bax (50 nM)-mediated release of 10-kDa CB-dextrans from the liposomes was determined as described in Supplemental Data. Data shown are means of 3 to 5 independent experiments with SD (error bars).
(F) Model of Bcl-2 conversion by NuBCP. Binding of NuBCP to the Bcl-2 loop displaces the BH4 domain, resulting in exposure of the BH3 domain, which leads to either release of pro-apoptotic BH3-only members (e.g., tBid) from Bcl-2 or inhibition of other anti-apoptotic relatives of Bcl-2 family (e.g., Bcl-XL) that leads to activation of pro-apoptotic multi-BH domain proteins (e.g., Bax).

Siva Kumar Kolluri, et al. Cancer Cell. ;14(4):285-298.
8.
Figure 6

Figure 6. NuBCP-9 induces Bcl-2-dependent Bax activation. From: A Short Nur77-derived Peptide Converts Bcl-2 from a Protector to a Killer.

(A) Bax dimerization/oligomerization. Isolated mitochondria from HeLa cells were incubated with purified Bax and/or Bcl-2 proteins preincubated with peptide (10 µM). After cross-linking with bismaleimidohexane (BMH), reactions were analyzed by immunoblotting using anti-Bax antibody (N20). Data from one of the five experiments are shown.
(B) Dose dependent activation of Bax. Isolated mitochondria were incubated with purified Bax and Bcl-2 proteins preincubated with NuBCP-9 (1, 2, and 5 µM) or tBid protein (100 ng), and analyzed for Bax activation as in (A). Levels of Hsp60 were used for control. Data from one of three experiments are shown.
(C) Bax dimerization/oligomerization in DoHH2 cells. Cells were treated with indicated peptide (10 µM) for 8 hr, and analyzed for Bax activation as in (A). Data from one of five experiments are shown.
(D) Inhibition of NuBCP-9-induced apoptosis by Bcl-2/1-95. DoHH2 cells transfected with DsRed-Bcl-2/1-95 or empty vector (pCMV-DsRed, Clontech) were exposed to the indicated peptide (10 µM) for 12 hr. Transfected cells were assessed for apoptosis by DAPI staining. Bars represent means ± SD from three experiments.
(E) Inhibition of NuBCP-9-induced Bax activation by Bcl-2/1-95. DoHH2 cells without transfection (left two panels) or transfected with DsRed-Bcl-2/1-95 (right four panels) were exposed to NuBCP-9 or NuBCP-9/AA (10 µM) as indicated for 12 hr. Cells were then immunostained with anti-Bax (6A7) antibody and analyzed by fluorescence microscopy. About 80% of nontransfected cells exposed to NuBCP-9 showed Bax staining (green) (left panels), while about 50% of DsRed-Bcl-2/1-95-transfected cells (red) failed to display the Bax staining (right panels). Scale, 10 µM.
(F) Bax activation in H460 cells. Cells treated with NuBCP-9 (6 µM) for 16 hr were immunostained with either polyclonal anti-Bax (Invitrogen) or monoclonal 6A7 anti-Bax antibody (Sigma) (Murphy et al., 2000) and anti-rabbit or anti-mouse SRPD-conjugated secondary antibodies. Immunofluorescence was analysed by flow cytometry. Histograms of peptide-treated cells (red) and non-treated cells (blue) were overlaid.
(G) Dominant-negative effect of Bcl-2 mutants. H460 cells in six-well plates were transfected with GFP (0.8 µg), empty vector (control), GFP-Bcl-2/1-90, or Bcl-2/ΔBH3 (2 µg) for 24 hr prior to exposure to NuBCP-9 (6 µM) for another 10 hr. Apoptosis of transfected cells was determined by Annexin V staining. Bars represent means ± SD from three experiments.
(H) Suppression of Bax activation by Bcl-2 mutants. Bcl-2 mutants were expressed and treated as in (G) and immunostained with anti-Bax (6A7) antibody and SRPD-conjugated secondary antibody. Bax immunoflourescence was analysed by flow cytometry. Fluorescence from transfected cells (green histogram) was compared to that from the non-transfected cells (red histogram) from the same transfection.
(I) NuBCP-9 induces Bax activation in MEFs but not Bcl-2−/−-MEFs. Cells were exposed to NuBCP-9 (10 µM) for 16 hr and immunostained with anti-Bax (6A7) antibody as in (F). Bax immunoflourescence was analysed by flow cytometry. Histograms of treated (red) and untreated (blue) cells were overlaid. Bax protein levels in MEF cells were determined by immunoblotting and did not change with NuBCP-9 treatment.
(J) Expression of Bcl-2 in Bcl-2−/−MEFs restores Bax activation by NuBCP. Bcl-2−/−MEFs in six-well plates were cotransfected with Bcl-2 (1 µg) and GFP or GFP-Nur77/478-504, (1 µg) and immunostained with anti-Bax (6A7) antibody. Bax immunoflourescence of GFP or GFP- Nur77/478-504 (GFP-NuBCP) expressing cells (purple histogram) were compared to Bcl-2 coexpressing cells (green histogram) from the same transfection by flow cytometry.

Siva Kumar Kolluri, et al. Cancer Cell. ;14(4):285-298.

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