Targeted mass spectrometry (LC/MS/MS) for Olaparib pharmacokinetics. For LC/MS/MS of Olaparib pharmacokinetics metabolites were extracted from
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1 Supplementary Methods: Targeted mass spectrometry (LC/MS/MS) for Olaparib pharmacokinetics For LC/MS/MS of Olaparib pharmacokinetics metabolites were extracted from mouse tumor samples and analyzed as previously described (1,2). Samples were measured using the Q1/Q3 transition of 435.2/281.3 for Olaparib. For the standard curve, Olaparib was prepared at 10-1 dilutions from 1mM to 1nM in 40% methanol. Lenti- and retroviral infection For infection, 2x10 6 /10-cm culture dish 293 packaging cells were plated and transfected using Lipofectamine 2000 (Invitrogen). 5x10 5 target cells/10-cm culture dish were infected with viral supernatants 48h-72h after transfection and selected with puromycin (2μg/ml, Sigma) LnCap cells were retroviral infected with: PRETRO Super shp53 (5 - GACTCCAGTGGTAATCTAC-3 ), pbabe-dominant-negative-p53 or empty vector and with psuper-shakt1 (5'- CCGGCGCGTGACCATGAACGAGTTTCTCGAGAAACTCGTTCATGGTCACGC GTTTTTG-3') and psuper-gfp as a control (3). Immunohistochemistry Sections were stained with γh2ax (cell signalling), anti-caspase-3 (cell signalling), cytokeratin 8 (Abcam) and 14 (Thermo Fisher) according to manufacture s recommendations. For quantification of stainings, three different 1
2 fields including tumor cells were quantified and the ratio of cells staining positive for caspase-3 was calculated. Supplementary Figure Legends: Supplementary Figure 1. Common genetic alterations in prostate cancer morph senescence into apoptosis in response to PARP inhibition. (A) Western Blot analysis of Pten Lx/Lx and Pten Lx/Lx ;Trp53 Lx/Lx MEFs to confirm successful excision of target genes upon Adeno-Cre infection. Western Blot analysis of Pten expression levels in Pten +/- MEFs. (B) SA-β-gal staining after Olaparib treatment of Pten +/-, (C) Pten Lx/Lx and Pten Lx/Lx ;Trp53 Lx/Lx MEFs. (D) Analysis of apoptosis in Pten Lx/Lx and Pten Lx/Lx ;Trp53 Lx/Lx MEFs by Annexin V staining upon treatment with 10uM Olaparib for 48h. (E) Quantification of apoptosis of (C) in MEFs of the indicated genotypes (* p<0.05). Supplementary Figure 2. PARP inhibition induces a differential response in vivo in CaP with a modest effect on overall tumor response. (A) SA-β-gal staining and quantification to visualize senescence in Pten +/- DLPs treated with vehicle (n=3) or Olaparib (n=3) (*p=0.0139). (B) Analysis and quantification of DNA damage in DLPs of Pten pc-/- mice upon Vehicle (n=3) or Olaparib (n=3) treatment (2 weeks) by γh2ax staining (*p=0.0238) (C) Analysis and quantification of DNA damage in DLPs of Pten pc-/- ;Trp53 pc-/- mice upon Vehicle (n=3) or Olaparib (n=3) treatment (1 week) by γh2ax staining (*p=0.0233) (D) 2
3 Analysis and quantification of apoptosis upon Olaparib treatment (1 week) by cleaved caspase 3 staining on DLPs derived from Pten pc-/- ;Trp53 pc-/- mice. Olaparib=50mg/kg Supplementary Figure 3. PARP inhibition induces a differential response in vivo in CaP with a modest effect on overall tumor response. (A) Analysis of luminal cells in DLPs of Pten pc-/- ;Trp53 pc-/- mice upon Vehicle (n=3) or Olaparib (n=3) treatment (1 week) by Cytokeratin 8 staining. (B) Analysis of intermediate basal cells in DLPs of Pten pc-/- ;Trp53 pc-/- mice upon Vehicle (n=3) or Olaparib (n=3) treatment (1 week) by Cytokeratin 14 staining. (C) LC-MS/MS analysis of Olaparib (50mg/kg) pharmacokinetics in different prostatic lobes. Supplementary Figure 4. Hyperactivation of the PI3K-Akt pathway impacts sensitivity towards Olaparib and the combination of PARP and PI3K inhibitors is effective in MEFs and human cancer cells. (A) Western Blot analysis of stable p53 knockdown in LnCap cells. (B) Western Blot analysis of stable overexpression of dominant negative p53 (DN) in LnCap cells. (C) Western Blot analysis of Akt1 knockdown in LnCap cells. (D) Quantification of growth inhibition by crystal violet staining of Pten Lx/Lx ;Trp53 Lx/Lx MEFs upon Olaparib, BKM-120 or combination treatment after 6 days. (E) Quantification of growth inhibition by crystal violet staining of DU145, (F) LnCap and (G) PC3 cells upon Olaparib, BKM-120 or combination treatment after 6 days. 3
4 Supplementary Figure 5. Combination of PARP and PI3K inhibitors as a novel therapeutic approach. (A) Schematic presentation of pre-clinical assessment of the in vivo efficacy of the combinatorial treatment of Olaparib and BKM-120 in 4 month old Pten pc-/- ;Trp53 pc-/- mice. (B) Representative MRI images of DLPs and (C) APs from Pten pc-/- ;Trp53 pc-/- mice demonstrating the assessment of tumor volume during treatment. Supplementary Figure 6. Combination of PARP and PI3K inhibitors as a novel therapeutic approach. (A) AP tumor volume change of Pten pc-/- ;Trp53 pc-/- mice upon vehicle (n=7), Olaparib (n=8), BKM-120 (n=6) or combination (n=9) treatment. (B) Analysis of luminal cells in DLPs of Pten pc-/- ;Trp53 pc-/- mice upon vehicle (n=3), Olaparib (n=3), BKM-120 (n=3) or combination (n=3) treatment by Cytokeratin 8 staining. (C) Analysis of intermediate basal cells in DLPs of Pten pc-/- ;Trp53 pc-/- mice upon vehicle (n=3), Olaparib (n=3), BKM-120 (n=3) or combination (n=3) treatment by Cytokeratin 14 staining. Olaparib=50mg/kg; BKM-120=30mg/kg Supplementary Figure 7. Combination of PARP and PI3K inhibitors as a novel therapeutic approach. (A) Progression-free survival in DLP tumors of Pten pc-/- ;Trp53 pc-/- mice upon vehicle (n=7), Olaparib (n=8), BKM-120 (n=6) or combination (n=9) treatment. (B) Progression-free survival in AP tumors of Pten pc-/- ;Trp53 pc-/- mice upon vehicle (n=7), Olaparib (n=8), BKM-120 (n=6) or 4
5 combination (n=9) treatment. (C) Overall survival of Pten pc-/- ;Trp53 pc-/- mice upon vehicle (n=7), Olaparib (n=8), BKM-120 (n=6) or combination (n=9) treatment. V=vehicle, O=Olaparib, B=BKM-120, O+B= combination Olaparib=50mg/kg; BKM-120=30mg/kg Supplementary References: 1. Juvekar A, Burga LN, Hu H, Lunsford EP, Ibrahim YH, Balmana J, et al. Combining a PI3K Inhibitor with a PARP Inhibitor Provides an Effective Therapy for BRCA1-Related Breast Cancer. Cancer Discovery. 2012;2: Yuan M, Breitkopf SB, Yang X, Asara JM. nprot Nat Protoc. Nature Publishing Group; 2012;7: Lin H-K, Chen Z, Wang G, Nardella C, Lee S-W, Chan C-H, et al. Skp2 targeting suppresses tumorigenesis by Arf-p53-independent cellular senescence. Nature. Nature Publishing Group; 2010;464:
Vulnerabilities of PTEN TP53 -Deficient Prostate Cancers to Compound PARP PI3K Inhibition
Downloaded from cancerdiscovery.aacrjournals.org on September 22, 218. 214 American Association for Cancer Published OnlineFirst May 27, 214; DOI: 1.1158/2159-829.CD-13-23 Vulnerabilities of PTEN TP53
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File Name: Supplementary Information Description: Supplementary Figures and Supplementary Table. File Name: Peer Review File Description:
File Name: Supplementary Information Description: Supplementary Figures and Supplementary Table File Name: Peer Review File Description: Supplementary Table 1 Primers and taqman probes used were the following:
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Supplemental Figure 1. Western blot analysis indicated that MIF was detected in the fractions of
Supplemental Figure Legends Supplemental Figure 1. Western blot analysis indicated that was detected in the fractions of plasma membrane and cytosol but not in nuclear fraction isolated from Pkd1 null
B16-F10 (Mus musculus skin melanoma), NCI-H460 (human non-small cell lung cancer
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LPS LPS P6 - + Supplementary Fig. 1.
P6 LPS - - - + + + - LPS + + - - P6 + Supplementary Fig. 1. Pharmacological inhibition of the JAK/STAT blocks LPS-induced HMGB1 nuclear translocation. RAW 267.4 cells were stimulated with LPS in the absence
Supplementary data Supplementary Figure 1 Supplementary Figure 2
Supplementary data Supplementary Figure 1 SPHK1 sirna increases RANKL-induced osteoclastogenesis in RAW264.7 cell culture. (A) RAW264.7 cells were transfected with oligocassettes containing SPHK1 sirna
To determine the effect of over-expression and/or ligand activation of. PPAR / on cell cycle, cell lines were cultured as described above until ~80%
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Soft Agar Assay. For each cell pool, 100,000 cells were resuspended in 0.35% (w/v)
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DOI: 10.1038/ncb3461 In the format provided by the authors and unedited. Supplementary Figure 1 (associated to Figure 1). Cpeb4 gene-targeted mice develop liver steatosis. a, Immunoblot displaying CPEB4
Supplementary Table; Supplementary Figures and legends S1-S21; Supplementary Materials and Methods
Silva et al. PTEN posttranslational inactivation and hyperactivation of the PI3K/Akt pathway sustain primary T cell leukemia viability Supplementary Table; Supplementary Figures and legends S1-S21; Supplementary
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Supplementary Information Supplementary Figure 1: cholesterol manipulation alters the positioning of autophagosomes in cells, related to figure 1. (a) HeLa cells were treated for 24h under conditions reducing
Expanded View Figures
MO reports PR3 dephosphorylates TZ Xian-o Lv et al xpanded View igures igure V1. PR3 dephosphorylates and inactivates YP/TZ., Overexpression of tight junction proteins Pals1 () or LIN7 () has no effect
Supporting Information
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Supplementary Figures Supplementary Figure S1. Binding of full-length OGT and deletion mutants to PIP strips (Echelon Biosciences). Supplementary Figure S2. Binding of the OGT (919-1036) fragments with
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