1. Volume 11, Issue 10, Pages 764-768 (May 2001)
Tumor suppressor and anti-inflammatory actions of PPARγ agonists are mediated via upregulation of PTEN 
Lisa Patel, Ian Pass, Phil Coxon, C.Peter Downes, Stephen A. Smith, Colin H. Macphee 
Current Biology 
Volume 11, Issue 10, Pages (May 2001)
DOI: /S (01)

2. Figure 1
PPARγ activation substantially increases PTEN mRNA, protein, and enzymic activity in human primary macrophages. Monocytes isolated from healthy donors were allowed to differentiate into macrophages over 4 days in the presence (filled bars) or absence (open bars) of 1 μM rosiglitazone. mRNA levels of osteopontin (a), PPARγ (b), MMP-9 (c), PTEN (d), and CD36 (e) were quantified on day 4 using the Taqman 5′ nuclease assay and normalized to levels found in freshly isolated monocytes to generate fold induction. (f) The effect of rosiglitazone concentration (1–1000 nM) on macrophage PTEN and SHIP2 mRNA levels. (g) Representative Western blot showing the concentration-dependent effects of rosiglitazone on macrophage PTEN, total PKB (T-PKB), and phosphorylated PKB (P-PKB) protein content. Western blots of total protein extracts were probed with mouse anti-PTEN antibodies (Santa Cruz Biotechnology) and then reprobed with sheep anti-phospho-PKB and anti-total PKB (Upstate Biotechnology) to confirm equal loading. (h) Representative experiment demonstrating enhanced macrophage PtdIns(3,4,5)P3 3-phosphatase activity in response to 1 μM rosiglitazone with accompanying PTEN/T-PKB Western blot (insert). PtdIns(3,4,5)P3 3-phosphatase activity assays were carried out on human macrophage lysates at 37°C for 5 min as described [23]. Data for (a–f) represent means ± SD from four different monocyte preparations. *p < 0.05 versus control
Current Biology  , DOI: ( /S (01) )

3. Figure 2
Specific PPARγ antisense oligonucleotides inhibit macrophage PTEN expression and influence cellular function. Freshly isolated human monocytes received either control (C) or antisense (AS) PPARγ oligonucleotides (designed and manufactured by Biognostik, Germany) at a final concentration of 1 μM and were incubated in culture for 7 days with medium changes every 2 to 3 days. The sequence lengths of antisense PPARγ (sequence ID: ) and the respective control (sequence ID: ) were the same, as were their G/C contents. mRNA levels of PPARγ (a), osteopontin (b), PTEN (c), and MMP-9 (f) were monitored at days 1, 4, and 7. (d) Western blot analysis of PPARγAS-treated macrophages demonstrating changes in PPARγ, PTEN, and P-PKB protein content but not PPARα, PPARδ, or T-PKB. PPARγ antibodies were purchased from Biomol GMBH, while PPARα and PPARδ antibodies were purchased from Affinity Bioreagents. (e) PPARγAS-mediated decrease in caspase 9 activity at day 7. Caspase activity was measured using a colorimetric apoptosis detection kit (R&D Systems). (g) Enhanced MMP-9 release by PPARγAS-treated macrophages. MMP-9 activity assays were performed on cell supernates using an MMP-9 ELISA kit (R&D Systems). Data are means ± SD from at least four different monocyte preparations except for the representative Western blot shown in (d). *p < 0.05, S versus AS
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4. Figure 3
PPARγ activation increases PTEN expression in both Caco2 and MCF7 cells, and reduces their rate of proliferation. Caco2 colorectal carcinoma (a) or MCF7 breast cancer (c) cells in a 96-well format were seeded at 1000 cells per well in 5% serum, and after 2 hr were monitored for zero time methyl[14C]thymidine incorporation as well as receiving 1 μM rosiglitazone or vehicle. Cellular thymidine incorporation was then followed at 12–24 hr intervals and the medium was changed every 2 days. Cell proliferation assays were carried out using Cytostar-T plates (Amersham Pharmacia) according to the manufacturer's instructions. (b) After a 96 hr incubation, Caco2 cells were lysed and PTEN, P-PKB, T-PKB, and p85α protein content were assessed by Western blot. PTEN and T-PKB protein were also measured after 48 hr for MCF7 cells (insert in [c]). *p < 0.01 versus control
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5. Figure 4
PPARγ binds two PPAR response elements in the genomic sequence upstream of PTEN. (a) The genomic sequence upstream of the PTEN transcription start site contains two putative PPARγ response elements. PPRE1 and PPRE2 correspond to the sequences spanning 6914 bp to 6953 bp and 8951 bp to 8990 bp of GenBank accession number AF067844, respectively. (b) Gel retardation analysis of in vitro-translated PPARγ. Radiolabeled PPRE1 and PPRE2 were incubated with nonprogrammed reticulocyte lysate (lanes 1 and 7, respectively) or in the presence of 1 or 5 μl of PPARγ-programmed reticulocyte lysate alone (lanes 2 and 3, and 8 and 9, respectively). 5 μl of programmed lysates was also incubated with a 100-fold molar excess of PPRE1 (lane 4) or PPRE2 (lane 10), or with a 100-fold excess of the DR1 element from the ApoCIII gene (lanes 5 and 11). Binding of PPARγ-programmed reticulocyte lysate to the DR1 PPAR response element from the ApoCIII gene promoter is shown in lanes 6 and 12. Protein-DNA complexes formed are indicated with an arrow. The [γ-32P]ATP probe was incubated for 20 min with the programmed/nonprogrammed reticulocyte lysate indicated, synthesized using the ProteinScript II kit (Ambion), before free and protein-bound DNA were separated on 6% DNA retardation gels (Invitrogen)
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