1. Volume 50, Issue 3, Pages 394-406 (May 2013)
Extracellular Adenosine Sensing—A Metabolic Cell Death Priming Mechanism Downstream of p53 
Jaclyn S. Long, Diane Crighton, James O’Prey, Gillian MacKay, Liang Zheng, Timothy M. Palmer, Eyal Gottlieb, Kevin M. Ryan 
Molecular Cell 
Volume 50, Issue 3, Pages (May 2013)
DOI: /j.molcel
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2. Molecular Cell 2013 50, 394-406DOI: (10.1016/j.molcel.2013.03.016)
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1 ADORA2B Is a p53 Target Gene
(A and B) TetOn-p53-WT or TetOn-p53-273H cells were treated with Dox for 24 hr prior to quantification of ADORA2B levels by qRT-PCR (A) and western blotting analysis of p53, p21, HDM2, and β-actin (B). Data in (A) are represented as mean ± SD.
(C) Immunofluorescence staining of A2B (green), p53 (red), and nucleus (DAPI, blue) in TetOn-p53-WT or TetOn-p53-273H cells treated with Dox for 48 hr.
(D) Saos-2 p53:ER cells were treated with or without 100 nM 4-hydroxytamoxifen (Tam) and/or 10 μg/ml cycloheximide (Chx) for 24 hr followed by qRT-PCR quantification of ADORA2B. Data are represented as mean ± SD.
(E) Sequence analysis of the ADORA2B gene revealed several potential p53 binding sites (i–v) within intron 1.
(F) Saos-2 cells were cotransfected with either an empty pcDNA3 vector or a wild-type-p53 (p53WT)-expressing construct along with luciferase reporter constructs containing potential binding sites i–v. Luciferase activity was analyzed 24 hr after transfection. Data are represented as mean ± SD.
(G) U2OS cells expressing scrambled (pRS Scr) or p53-specific (pRS p53) shRNAs were treated with 2 nM actinomycin D (ActD), 10 μM nutlin, 20 μM cisplatin, 2 μM methotrexate (MTX), or 100 J/m2 UV for 16 hr prior to qRT-PCR quantification of ADORA2B. Data are represented as mean ± SD. See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 A2B Activation Promotes Apoptosis
(A) WT and p53−/− MEF E1a-ER cells were incubated with or without 500 nM Tam for 8 hr followed by 10 μM cisplatin for a further 24 hr prior to qRT-PCR analysis of ADORA2B. Data are represented as mean ± SD.
(B) Saos-2 cells transfected with either pcDNA3, pcDNA3-myc-ADORA2B (myc-ADORA2B), or pcDNA3-p53-WT (p53WT) and CD20 (as a transfection marker) were treated with or without NECA for 48 hr. Sub-G1 content was analyzed in the CD20-positive population by flow cytometry. Data are represented as mean ± SD.
(C–G) TetOn-A2B Saos-2 cells were treated with or without Dox, NECA, zVAD-fmk (75 μM), and 2 μg/ml TNF-α + 4 μg/ml cycloheximide (CHX) where indicated for 48 hr prior to analysis for sub-G1 content by flow cytometry (C), TUNEL staining (D), and western blotting detection of PARP (E), cleaved caspases 3, 7, and 9 (F), and cleaved caspase 8 (G). Data in (C) are represented as mean ± SD. See also Figure S2.
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5. Figure 3 A2B Contributes to p53-Induced Cell Death
(A and B) TetOn-p53-WT cells were treated with or without Dox and/or NECA for 30 hr prior to measurement of sub-G1 DNA content using flow cytometry (A) or western blotting for p53 and β-actin (B). Data in (A) are represented as mean ± SD.
(C) TetOn-p53-WT cells were transfected with either nonsilencing (NS) or three different ADORA2B-specific (A1–A3) siRNAs. At 48 hr after transfection, the cells were treated with or without Dox + NECA for a further 24 hr prior to qRT-PCR analysis of ADORA2B. Data are represented as mean ± SD.
(D) TetOn-p53-WT cells were transfected with either nonsilencing (NS) or three different ADORA2B-specific (A1–A3) siRNAs. At 48 hr after transfection, the cells were treated with or without Dox + NECA for a further 24 hr prior to analysis of sub-G1-DNA content. Data are represented as mean ± SD.
(E) TetOn-p53-WT cells were transfected with either nonsilencing (NS) or three different ADORA2B-specific (A1–A3) siRNAs. At 48 hr after transfection, the cells were treated with or without Dox for a further 24 hr prior to qRT-PCR analysis of ADORA2B. Data are represented as mean ± SD.
(F) TetOn-p53-WT cells were transfected with either nonsilencing (NS) or three different ADORA2B-specific (A1–A3) siRNAs. At 48 hr after transfection, the cells were treated with or without Dox for a further 24 hr prior to analysis of sub-G1-DNA content. Data are represented as mean ± SD. See also Figure S3.
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6. Figure 4 A2B Negatively Regulates Bcl-2 and Bcl-XL
(A–C) TetOn-A2B Saos-2 cells were treated with or without Dox and/or NECA for 48 hr prior to western blotting analysis of Bak and Bax (A), Bcl-2 (B), and Bcl-XL (C).
(D and E) TetOn-A2B Saos-2 cells were pretreated with or without zVAD-fmk (75 μM) for 1 hr followed by Dox and/or NECA treatment, where indicated, for 48 hr prior to western blotting for Bcl-2 (D) and Bcl-XL (E).
(F) Wild-type Saos-2 cells were treated with 25 μM ABT-737 for 48 hr before being harvested for flow cytometry analysis of sub-G1 DNA content. Data are represented as mean ± SD.
(G and H) TetOn-A2B control and Bcl-2- or Bcl-XL-overexpressing Saos-2 cells were treated with or without Dox and/or NECA for 48 hr prior to flow cytometry analysis of sub-G1 DNA content or western blotting for Bcl-2, Bcl-XL, and β-actin. Data are represented as mean ± SD.
(I) TetOn-p53-WT cells were treated with or without Dox and/or NECA in the presence of zVAD-fmk (75 μM) for 48 hr prior to western blotting analysis of Bcl-2, Bcl-XL, and total p42. See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5 A2B-Induced Death Is Puma Dependent
(A) Levels of BH3-only proteins Puma, Noxa, Hrk, Bim, Bik, and Bad following Dox and/or NECA treatment of TetOn-A2B cells for 48 hr were analyzed by western blotting.
(B) qRT-PCR analysis confirming overexpression of myc-A2B in wild-type and puma−/− HCT116 A2B cells. Data are represented as mean ± SD.
(C) Wild-type HCT116 cells expressing control vector or myc-A2B were treated with or without NECA for 48 hr prior to analysis of sub-G1 DNA content by flow cytometry.
(D) Puma−/− HCT116 cells expressing control vector or myc-A2B were treated with or without NECA for 48 hr prior to analysis of sub-G1 DNA content by flow cytometry. Data are represented as mean ± SD. See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Hypoxia Promotes Extracellular Adenosine Accumulation that Stimulates A2B-Dependent Cell Death
(A) HCT116 control and HCT116.A2B cells were incubated in either normoxic (21% O2) or hypoxic (1% O2) conditions for 48 hr prior to flow cytometry analysis of sub-G1 DNA content. Data are represented as mean ± SD.
(B) Extracellular adenosine detection by LC-MS analysis of media obtained from HCT116 control cells incubated in either normoxic (21% O2) or hypoxic (1% O2) conditions for 36 hr. Data are represented as mean ± SD.
(C and D) HCT116 control and HCT116.A2B cells were pretreated with zVAD-fmk (75 μM) (C) or PSB603 (2 μM) (D) for 1 hr prior to incubation in either normoxic (21% O2) or hypoxic (1% O2) conditions for 48 hr. Cells were subsequently harvested and analyzed for sub-G1 DNA content by flow cytometry. Data are represented as mean ± SD.
(E) The involvement of puma in A2B-mediated cell death was examined in wild-type and puma−/− HCT116 cells expressing myc-A2B grown in normoxic (21% O2) or hypoxic (1% O2) conditions for 48 hr. Data are represented as mean ± SD.
(F) Levels of Bcl-2, Bcl-XL, Mcl-1, and HSP90 in HCT116 control and HCT116.A2B cells grown in normoxic or hypoxic conditions for 48 hr were analyzed by western blotting. See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7 A2B Signaling Contributes to Chemotherapy-Induced Death
(A) The effect of 2 nM Act D or 20 μM cisplatin (48 hr) on cell-cycle profile of U2OS E1a cells was examined by flow cytometry.
(B) Extracellular adenosine measurement by LC-MS analysis of media obtained from U2OS cells incubated with or without 2 nM Act D or 20 μM cisplatin for 48 hr. Data are represented as mean ± SD.
(C) Effect of PSB603 (2 μM) on cell death in U2OS E1a cells treated with 20 μM cisplatin for 48 hr as examined by flow cytometry. Data are represented as mean ± SD.
(D) Extracellular adenosine measurement by LC-MS analysis of media obtained from MEF E1a-ER cells pretreated with zVAD-fmk prior to incubation with 500 nM Tam for 8 hr followed by 10 μM cisplatin for an additional 24 hr. Data are represented as mean ± SD.
(E) Effect of PSB603 (2 μM) on cell death in MEF E1a-ER cells treated with 500 nM Tam for 8 hr followed by 10 μM cisplatin for an additional 24 hr as examined by flow cytometry. See also Figure S7. Data are represented as mean ± SD.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions