Journal:

Article Title: Peroxisome Proliferator-Activated Receptor-γ Regulates the Expression of Alveolar Macrophage Macrophage Colony-Stimulating Factor

doi:

Figure Lengend Snippet: PPARγ knockout alveolar macrophage Pathology resembles the GM-CSF KO model. GM-CSF KO (A), PPARγ KO (B), and C57BL/6 control (C) mice were sacrificed, and the lungs stained with periodic-acid Schiff (×40). The GM-CSF KO model demonstrates scattered foci of intraalveolar PAS-positive material (thick arrows, A) and lipid-engorged alveolar macrophages (thick arrow, ×100 inset). All of the PPARγ KO mice (7 of 7 mice) exhibited similar PAS-positive material in alveolar macrophages (B, ×100 inset). Similar to the GM-CSF KO animals, several of these mice (2 of 7) also developed intraalveolar PAS-positive material (B, thin arrow).

Article Snippet: Primary cells were allowed to adjust to culture overnight before the addition of stimuli: LPS (50 –500 ng/ml), M-CSF (100 ng/ml), irreversible PPARγ antagonist GW9662 (10 μM; Sigma-Aldrich), and/or PPARγ agonist pioglitazone (10 μM, Takeda Chemical Industries).

Techniques: Knock-Out, Control, Staining

Journal:

Article Title: Peroxisome Proliferator-Activated Receptor-γ Regulates the Expression of Alveolar Macrophage Macrophage Colony-Stimulating Factor

doi:

Figure Lengend Snippet: Cell counts in BAL

Article Snippet: Primary cells were allowed to adjust to culture overnight before the addition of stimuli: LPS (50 –500 ng/ml), M-CSF (100 ng/ml), irreversible PPARγ antagonist GW9662 (10 μM; Sigma-Aldrich), and/or PPARγ agonist pioglitazone (10 μM, Takeda Chemical Industries).

Techniques: Cell Counting

Journal:

Article Title: Peroxisome Proliferator-Activated Receptor-γ Regulates the Expression of Alveolar Macrophage Macrophage Colony-Stimulating Factor

doi:

Figure Lengend Snippet: M-CSF gene expression and protein are elevated in BAL specimens from GM-CSF and PPARγ KO mice. Acellular BAL fluid was assessed for M-CSF protein by ELISA, and BAL cell pellets were evaluated for M-CSF mRNA. Compared with wild-type mice (n = 5), BAL cells from both GMCSF KO (n = 4) and PPARγ KO (n = 4) mice demonstrated increased M-CSF gene expression (p ≤ 0.002 for each comparison, B). Similarly, BAL protein levels were increased in both models when compared with wild-type mice (B, p < 0.002, n = 4 per condition).

Article Snippet: Primary cells were allowed to adjust to culture overnight before the addition of stimuli: LPS (50 –500 ng/ml), M-CSF (100 ng/ml), irreversible PPARγ antagonist GW9662 (10 μM; Sigma-Aldrich), and/or PPARγ agonist pioglitazone (10 μM, Takeda Chemical Industries).

Techniques: Gene Expression, Enzyme-linked Immunosorbent Assay, Comparison

Journal:

Article Title: Peroxisome Proliferator-Activated Receptor-γ Regulates the Expression of Alveolar Macrophage Macrophage Colony-Stimulating Factor

doi:

Figure Lengend Snippet: Transfection of PPARγ expression vector into RAW 264.7 cells attenuates LPS-mediated induction of M-CSF. RAW 264.7 cells were transfected with 1 μg PPARγ murine expression vector. We measured the expression of M-CSF after 24 h in the presence and absence of LPS. Transient transfection of the PPARγ expression vector resulted in 10,809 ± 1,578% (mean ± SEM) increased expression of PPARγ mRNA expression compared with the control (β-gal) vector (right bars, n = 3, p ≤ 0.001). Compared with transfected controls (β-gal), LPS-induced M-CSF production was inhibited by 90.1 ± 13.2% with the overexpression of PPARγ expression vector (left bars, p = 0.017, n = 3).

Article Snippet: Primary cells were allowed to adjust to culture overnight before the addition of stimuli: LPS (50 –500 ng/ml), M-CSF (100 ng/ml), irreversible PPARγ antagonist GW9662 (10 μM; Sigma-Aldrich), and/or PPARγ agonist pioglitazone (10 μM, Takeda Chemical Industries).

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Over Expression

Journal:

Article Title: Peroxisome Proliferator-Activated Receptor-γ Regulates the Expression of Alveolar Macrophage Macrophage Colony-Stimulating Factor

doi:

Figure Lengend Snippet: M-CSF secretion is enhanced by PPARγ antagonists and suppressed by PPARγ agonist. RAW cells were cultured in the presence and absence of pioglitazone or GW9662 at baseline or after stimulation with LPS. LPS-stimulated RAW cells treated with pioglitazone expressed 41 ± 6% significantly less M-CSF mRNA (A, p = 0.04, n = 4) and 12 ± 0.7% less M-CSF protein (p = 0.01) compared with the LPS cultures alone. Conversely, baseline RAW cells treated with GW9662 increased their M-CSF gene expression and protein by 532 ± 242% (B, p = 0.02, n = 3) and 11.2 ± 1% (p = 0.03), respectively.

Article Snippet: Primary cells were allowed to adjust to culture overnight before the addition of stimuli: LPS (50 –500 ng/ml), M-CSF (100 ng/ml), irreversible PPARγ antagonist GW9662 (10 μM; Sigma-Aldrich), and/or PPARγ agonist pioglitazone (10 μM, Takeda Chemical Industries).

Techniques: Cell Culture, Gene Expression

Journal:

Article Title: Peroxisome Proliferator-Activated Receptor-γ Regulates the Expression of Alveolar Macrophage Macrophage Colony-Stimulating Factor

doi:

Figure Lengend Snippet: PPARγ regulates M-CSF through NF-κB. RAW 264.7 cells were stimulated with LPS, which resulted in a 21.6 ± 0.9-fold increase in M-CSF expression. LPS induced M-CSF expression was reduced by 60 ± 26% when the NF-κB was inhibited with the proteasomal blocker. Pioglitazone treatment also decreased LPS-stimulated M-CSF expression, by 68 ± 15% of the LPS control (p < 0.05 vs LPS alone). The combination of both pioglitazone and NF-κB inhibition resulted in 80 ± 25% reduction of M-CSF expression (p < 0.001 vs LPS). These data suggest that PPARγ can regulate M-CSF induction through repression of NF-κB.

Article Snippet: Primary cells were allowed to adjust to culture overnight before the addition of stimuli: LPS (50 –500 ng/ml), M-CSF (100 ng/ml), irreversible PPARγ antagonist GW9662 (10 μM; Sigma-Aldrich), and/or PPARγ agonist pioglitazone (10 μM, Takeda Chemical Industries).

Techniques: Expressing, Control, Inhibition

Journal:

Article Title: Peroxisome Proliferator-Activated Receptor-γ Regulates the Expression of Alveolar Macrophage Macrophage Colony-Stimulating Factor

doi:

Figure Lengend Snippet: Pioglitazone inhibits LPS-mediated NF-κB activation. Whole-cell extracts were analyzed by EMSA using a 32P-labeled oligonucleotide containing the κB consensus sequences and analyzed on a 4% nondenaturing acrylamide gel. Specificity of the band was confirmed by supershift analysis with p50 and p65 Abs and competition with cold oligonucleotide. Representative image from four independent experiments is shown. Quantification using the StormImager with ImageQuant analysis revealed that pretreatment with pioglitazone before addition of LPS decreased NF-κB binding by 30.6 ± 6.9% (mean (SEM), p < 0.01 by Tukey's test). US, Unstimulated; Pio, pioglitazone.

Article Snippet: Primary cells were allowed to adjust to culture overnight before the addition of stimuli: LPS (50 –500 ng/ml), M-CSF (100 ng/ml), irreversible PPARγ antagonist GW9662 (10 μM; Sigma-Aldrich), and/or PPARγ agonist pioglitazone (10 μM, Takeda Chemical Industries).

Techniques: Activation Assay, Labeling, Acrylamide Gel Assay, Binding Assay

Journal:

Article Title: Peroxisome Proliferator-Activated Receptor-γ Regulates the Expression of Alveolar Macrophage Macrophage Colony-Stimulating Factor

doi:

Figure Lengend Snippet: PPARγ inhibits LPS-induced NF-κB association with the M-CSF promoter. ChIP was performed using BALB/c bone marrow-derived macrophages. After treatment with LPS (2 μg/ml), pioglitazone (Pio, 10 μg/ml), or GW9662 (GW, 10 mM), cell lysates were subjected to immunoprecipitation with anti-PPARγ, anti-c-Rel, or anti-p65. Binding to the M-CSF promoter was determined by real-time PCR. LPS stimulation resulted in c-Rel and p65 binding to the NF-κB site upstream of the M-CSF promoter (B). Activation of PPARγ by pioglitazone increased PPARγ association with the NF-κB response element, which was nearly eliminated with NF-κB activation by LPS (A). Pioglitazone also resulted in PPARγ binding to the M-CSF PPRE promoter region (C).

Article Snippet: Primary cells were allowed to adjust to culture overnight before the addition of stimuli: LPS (50 –500 ng/ml), M-CSF (100 ng/ml), irreversible PPARγ antagonist GW9662 (10 μM; Sigma-Aldrich), and/or PPARγ agonist pioglitazone (10 μM, Takeda Chemical Industries).

Techniques: Derivative Assay, Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Activation Assay

Journal:

Article Title: Peroxisome Proliferator-Activated Receptor-γ Regulates the Expression of Alveolar Macrophage Macrophage Colony-Stimulating Factor

doi:

Figure Lengend Snippet: Model of PPARγ regulation of M-CSF transcription. Data from the ChIP and culture studies were put together to develop a model of a working hypothesis in which PPARγ regulates M-CSF through NF-κB. PPARγ inhibits association of NF-κB proteins with the promoter, an effect that is enhanced in the presence of PPARγ activation. In the presence of NF-κB activation, PPARγ binding to the promoter is down-regulated unless PPARγ is ligand-activated by pioglitazone. In the absence of PPARγ, there is a potential constitutive production of M-CSF through p65/c-Rel binding to NF-κB transcription site.

Article Snippet: Primary cells were allowed to adjust to culture overnight before the addition of stimuli: LPS (50 –500 ng/ml), M-CSF (100 ng/ml), irreversible PPARγ antagonist GW9662 (10 μM; Sigma-Aldrich), and/or PPARγ agonist pioglitazone (10 μM, Takeda Chemical Industries).

Techniques: Activation Assay, Binding Assay

Journal: PLoS ONE

Article Title: Quercetin Inhibits IL-1β-Induced Inflammation, Hyaluronan Production and Adipogenesis in Orbital Fibroblasts from Graves' Orbitopathy

doi: 10.1371/journal.pone.0026261

Figure Lengend Snippet: (A) Orbital fibroblasts (1×10 5 ) of normal and Graves' orbitopathy (GO) patients were seeded into 24-well culture plates and treated with different concentrations of quercetin (10, 30, 50, or 100 µM) for 24 h. After treatment, assays with 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) were performed to test for viability. (B) An annexin V/FITC kit was used to detect phosphatidylserine externalization, as an index of apoptosis. Percentage of stained cells with annexin V was analyzed by flow cytometry. (C) Orbital fibroblasts (1×10 5 ) of GO patients were seeded into 24-well culture plates and treated with different concentrations of quercetin (10, 50, 100, or 200 µM) for 3 days in adipogenic medium containing adipogenesis inducers and rosiglitazone (10 µM). After treatment, MTT assays were performed. Results are expressed as percentage of untreated control values presented as mean ± standard deviation (SD). Assays were performed at least three times in triplicate; data from a representative experiment are shown, expressed as the differences between treated and untreated cells.

Article Snippet: A PPARγ agonist, rosiglitazone (10 μM, Cayman, Ann Arbor, MI, USA), was added from day 1 for further stimulation of adipogenesis.

Techniques: Staining, Flow Cytometry, Control, Standard Deviation

Journal: PLoS ONE

Article Title: Quercetin Inhibits IL-1β-Induced Inflammation, Hyaluronan Production and Adipogenesis in Orbital Fibroblasts from Graves' Orbitopathy

doi: 10.1371/journal.pone.0026261

Figure Lengend Snippet: (A–B) Quercetin (50 or 100 µM) treatment for the first 3 days after initiation of 10-day adipogenesis in adipogenic media containing (A) 10 µM rosiglitazone, or (B) combined 10 µM rosiglitazone and 10 ng/ml IL-1β. Cells were stained with Oil Red O and examined grossly and microscopically (×40; inset ×400). (C) Cell-bound Oil Red O was solubilized and optical density (OD) read at 490 nm to obtain a quantitative assessment of adipogenesis. The experiments were performed in triplicate with cells from three different donors, and data in the column are the mean relative density ratios ± SD of three experiments. * P <0.001 vs. untreated control differentiated cells.

Article Snippet: A PPARγ agonist, rosiglitazone (10 μM, Cayman, Ann Arbor, MI, USA), was added from day 1 for further stimulation of adipogenesis.

Techniques: Staining, Control

Journal: PLoS ONE

Article Title: Quercetin Inhibits IL-1β-Induced Inflammation, Hyaluronan Production and Adipogenesis in Orbital Fibroblasts from Graves' Orbitopathy

doi: 10.1371/journal.pone.0026261

Figure Lengend Snippet: (A) Quercetin (50 or 100 µM) treatment for the first 3 days after initiation of 10-day adipogenesis in adipogenic media containing 10 µM rosiglitazone, or combined 10 µM rosiglitazone and 10 ng/ml IL-1β. After 10 days, cell lysates were subjected to western blot analysis of PPARγ, C/EBPα, and C/EBPβ protein expression. The experiments were performed in triplicate with cells from three different donors. (B–D) Quantification by densitometry, normalized to the β-actin level in the same sample, is shown for PPARγ (B), C/EBPα (C), and C/EBPβ (D). The data in the column are the mean relative density ratios ± SD of three experiments. * P <0.05, ** P <0.001 vs. untreated control differentiated cells.

Article Snippet: A PPARγ agonist, rosiglitazone (10 μM, Cayman, Ann Arbor, MI, USA), was added from day 1 for further stimulation of adipogenesis.

Techniques: Western Blot, Expressing, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Peroxisome proliferator-activated receptor-γ enhances human pulmonary artery smooth muscle cell apoptosis through microRNA-21 and programmed cell death 4

doi: 10.1152/ajplung.00532.2016

Figure Lengend Snippet: PDCD4 levels and apoptosis in peroxisome proliferator-activated receptor-γ (PPARγ)-depleted HPASMCs. HPASMCs were transfected with nontargeting scrambled oligonucleotides or PPARγ (20 nM) dicer-substrate short interfering (si)RNA constructs for the first 6 h of 48- or 72-h incubation periods. A: PPARγ protein expression in HPASMCs transfected with PPARγ siRNA. B: PDCD4 mRNA levels in HPASMCs transfected with PPARγ siRNA. C: PDCD4 protein levels in HPASMCs transfected with PPARγ siRNA. D: caspase-3 activity in HPASMCs transfected with nontargeting scrambled oligonucleotides or siPPARγ. E: cytochrome-c levels were analyzed using an ELISA on HPASMCs transfected with nontargeting scrambled (SCR) oligonucleotides or siPPARγ. A and C: representative immunoblots. Bars represent means ± SE for PPARγ, PDCD4, caspase-3 activity, or cytochrome-c abundance; n = 6–7. **P < 0.01 vs. SCR; ***P < 0.001 vs. scrambled (SCR; ****P < 0.0001 vs. SCR.

Article Snippet: Selected HPASMC monolayers were then placed in a normobaric hypoxia chamber (1% O 2 -5% CO 2 ; Biospherix, Lacuna, NY) or propagated in a cell culture incubator under normoxic conditions (21% O 2 -5% CO 2 ) for 48–72 h. In selected studies, to activate PPARγ, the pharmacological PPARγ agonist RSG (10–25 μM) or an equal volume of dimethyl sulfoxide (DMSO; 1%; Fisher Scientific, Fair Lawn, NJ) vehicle was added to the HPASMC culture media.

Techniques: Transfection, Construct, Incubation, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Peroxisome proliferator-activated receptor-γ enhances human pulmonary artery smooth muscle cell apoptosis through microRNA-21 and programmed cell death 4

doi: 10.1152/ajplung.00532.2016

Figure Lengend Snippet: Time- and dose-dependent effects of PPARγ ± rosiglitazone (RSG) on PDCD4 and PPARγ protein expression. HPASMC monolayers were propagated in a cell culture incubator at 37◦ C until reaching 60–80% confluence. Selected HPASMCs were transfected with AdGFP [25 multiplicity of infection (MOI)] or escalating doses of AdPPARγ (5–25 MOI) for 6 h. After 24 h, RSG (10 μM) or an equal volume of DMSO was added to the cell culture media. Following the conclusion of 48- or 72-h incubation periods, total protein was isolated from monolayer lysates. A and B: PPARγ or PDCD4 protein levels were detected in HPASMCs transfected with AdGFP or AdPPARγ ± RSG for 48 h. C: full-length representative immunoblots are shown to demonstrate PPARγ, PDCD4 and GAPDH proteins in HPASMCs transfected with AdGFP or AdPPARγ + RSG for 48 h. D and E: PPARγ or PDCD4 protein levels were detected in HPASMCs transfected with AdGFP or AdPPARγ ± RSG for 72 h. Representative immunoblots are displayed in A, B, D, and E; n = 6. *P < 0.05 vs. AdGFP (−RSG); ***P < 0.001 vs. AdGFP (−RSG); ****P < 0.0001 vs. AdGFP (−RSG); #P < 0.05 vs. AdGFP (+RSG); ##P < 0.01 vs. AdGFP (+RSG); ###P < 0.001 vs. AdGFP (+RSG); ####P < 0.0001 vs. AdGFP (+RSG).

Article Snippet: Selected HPASMC monolayers were then placed in a normobaric hypoxia chamber (1% O 2 -5% CO 2 ; Biospherix, Lacuna, NY) or propagated in a cell culture incubator under normoxic conditions (21% O 2 -5% CO 2 ) for 48–72 h. In selected studies, to activate PPARγ, the pharmacological PPARγ agonist RSG (10–25 μM) or an equal volume of dimethyl sulfoxide (DMSO; 1%; Fisher Scientific, Fair Lawn, NJ) vehicle was added to the HPASMC culture media.

Techniques: Expressing, Cell Culture, Transfection, Infection, Incubation, Isolation, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Peroxisome proliferator-activated receptor-γ enhances human pulmonary artery smooth muscle cell apoptosis through microRNA-21 and programmed cell death 4

doi: 10.1152/ajplung.00532.2016

Figure Lengend Snippet: PPARγ modulates PDCD4 protein, HPASMC proliferation, and apoptosis in response to hypoxia. HPASMC monolayers were exposed to normoxic (21% O2) or hypoxic (1% O2) conditions for 72 h. At the onset of the study, selected HPASMCs were transfected with AdGFP (10 MOI) or AdPPARγ (10 MOI) for 6 h. RSG (10 μM) was added to the culture medium after 24 h of incubation. Upon the conclusion of the study period, total protein was extracted from HPASMC monolayer lysates, and proliferation and apoptosis assays were conducted. A: demonstration of PDCD4 protein expression in HPASMCs with representative immunoblot. B: proliferation was detected in HPASMCs using automated cell counting. C: demonstration of caspase-3 activation as an indicator of apoptosis in normoxia- or hypoxia-exposed HPASMCs treated with AdPPARγ or AdGFP + RSG. D: HPASMCs exposed to normoxia or hypoxia for 72 h were treated with RSG (10–25 μM) or an equal volume of DMSO at the onset of the study period. With the use of FACS analysis, apoptosis was examined by detecting annexin V staining in HPASMCs. Staurosporine (0.1 μM) was applied to selected normoxia-exposed HPASMCs as a positive control to enhance apoptosis. Bars represent means ± SE for PDCD4 protein (A), proliferation (B), caspase-3 activation (C), or annexin V (D) detection by median fluorescence. A: n = 12; ****P < 0.0001 vs. NOR(−); $P < 0.05 vs. NOR (+); ++P < 0.01 vs. HYP(−). B: n = 3; and C: n = 6. *P < 0.05 vs. NOR(−); **P < 0.01 vs. NOR(−); +++P < 0.001 vs. HYP(−). D: n = 3; ***P < 0.001 vs. NOR (DMSO); ****P < 0.0001 vs. NOR (DMSO); ++++P < 0.0001 vs. NOR (10 μM); ###P < 0.001 vs. HYP (DMSO); ####P < 0.0001 vs. HYP (DMSO); $$$P < 0.001 vs. HYP (10 μM).

Article Snippet: Selected HPASMC monolayers were then placed in a normobaric hypoxia chamber (1% O 2 -5% CO 2 ; Biospherix, Lacuna, NY) or propagated in a cell culture incubator under normoxic conditions (21% O 2 -5% CO 2 ) for 48–72 h. In selected studies, to activate PPARγ, the pharmacological PPARγ agonist RSG (10–25 μM) or an equal volume of dimethyl sulfoxide (DMSO; 1%; Fisher Scientific, Fair Lawn, NJ) vehicle was added to the HPASMC culture media.

Techniques: Transfection, Incubation, Expressing, Western Blot, Cell Counting, Activation Assay, Staining, Positive Control, Fluorescence

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Peroxisome proliferator-activated receptor-γ enhances human pulmonary artery smooth muscle cell apoptosis through microRNA-21 and programmed cell death 4

doi: 10.1152/ajplung.00532.2016

Figure Lengend Snippet: Effects of PPARγ overexpression on PDCD4 expression, proliferation, and apoptosis in PDCD4-depleted HPASMCs. PDCD4 levels, proliferation, and apoptosis were measured in PDCD4-depleted HPASMCs in which PPARγ was overexpressed. HPASMCs were transfected with AdGFP or AdPPARγ for 6 h at the onset of the experiment. After 24 h of incubation, RSG (10 μM) was added to all HPASMC monolayers, and selected HPASMCs were transfected with nontargeting scrambled oligonucleotides or siPDCD4 (20 nM). Upon the conclusion of the 72-h study period, total protein was extracted from HPASMC monolayer lysates, and proliferation and apoptosis assays were conducted. A: demonstration of PDCD4 protein expression in HPASMCs with representative immunoblot. B: proliferation was measured in HPASMCs using automated cell counting. C: demonstration of caspase-3 activation as an indicator of apoptosis in HPASMCs. Bars represent means ± SE for PDCD4 protein (A), HPASMC proliferation (B), or caspase-3 cleavage (C). A and C: n = 3; *P < 0.05 vs. SCR(−); **P < 0.01 vs. SCR(−); ++P < 0.01 vs. siPDCD4(−). B: n = 7; ****P < 0.0001 vs. SCR(−); ####P < 0.0001 vs. siPDCD4 (−).

Article Snippet: Selected HPASMC monolayers were then placed in a normobaric hypoxia chamber (1% O 2 -5% CO 2 ; Biospherix, Lacuna, NY) or propagated in a cell culture incubator under normoxic conditions (21% O 2 -5% CO 2 ) for 48–72 h. In selected studies, to activate PPARγ, the pharmacological PPARγ agonist RSG (10–25 μM) or an equal volume of dimethyl sulfoxide (DMSO; 1%; Fisher Scientific, Fair Lawn, NJ) vehicle was added to the HPASMC culture media.

Techniques: Over Expression, Expressing, Transfection, Incubation, Western Blot, Cell Counting, Activation Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Peroxisome proliferator-activated receptor-γ enhances human pulmonary artery smooth muscle cell apoptosis through microRNA-21 and programmed cell death 4

doi: 10.1152/ajplung.00532.2016

Figure Lengend Snippet: Effects of PPARγ on miR-21 expression and HPASMC proliferation and apoptosis. In selected studies, PPARγ was depleted by transfecting HPASMCs with siPPARγ (20 nM). A: demonstration of the expression of miR-21 in PPARγ-depleted HPASMCs compared with HPASMCs transfected with nontargeting scrambled oligonucleotides (20 nM). B: demonstration of the functional effects of PPARγ on HPASMC proliferation in PPARγ-depleted HPASMCs compared with HPASMCs transfected with nontargeting scrambled oligonucleotides. The effects of gain or loss of miR-21 expression on PDCD4 protein levels were examined. C: PDCD4 protein levels were measured in HPASMCs transfected with mature miR-21 mimics (30 nM) or nontargeting scrambled oligonucleotides (30 nM) with a representative immunoblot. D: PDCD4 protein levels were measured in HPASMCs exposed to normoxic (21% O2) or hypoxic (1% O2) conditions for 72 h. Six hours before the cells were placed in normoxic or hypoxic conditions, HPASMC monolayers were transfected with locked nucleic acid siRNA sequences targeting miR-21 (anti-miR-21; 1 nM) or nontargeting control locked nucleic acid scrambled RNA sequences (1 nM). Representative immunoblot is shown. E: miR-21 expression was measured in HPASMC monolayers exposed to normoxic (21% O2) or hypoxic (1% O2) conditions. Selected HPASMCs were transfected with AdGFP (10 MOI) or AdPPARγ (10 MOI) for 6 h at the onset of the study. RSG (10 μM) was added to the culture medium after 24 h of incubation and miR-21 levels were detected using quantitative RT-PCR upon the conclusion of the 72-h study. F and G: effects of miR-21 overexpression on apoptosis and proliferation in HPASMCs were examined. HPASMCs were transfected with AdGFP (10 MOI) or AdPPARγ (10 MOI) for 6 h at the onset of the study. After 24 h of incubation, RSG (10 μM) was added to all HPASMC monolayers and selected HPASMCs were transfected with mature miR-21 mimics (30 nM) or nontargeting scrambled oligonucleotides (30 nM). F: demonstration of the spectrophotometric detection of absorbance at 405 nm reflecting caspase-3 activity as an indicator of apoptosis. G: proliferation was measured in HPASMCs using automated cell counting. A and B: n = 3; *P < 0.05 vs. SCR. C: n = 9; ****P < 0.0001 vs. SCR. D: n = 9 and E: n = 3; **P < 0.01 vs. NOR(−); ***P < 0.001 vs. NOR(−); +P < 0.05 vs. HYP(−); ++P < 0.01 vs. HYP(−). F and G: n = 5–6; **P < 0.01 vs. SCR(−); ***P < 0.001 vs. SCR(−); ****P < 0.0001 vs. SCR(−); ++P < 0.01 vs. miR-21(−); ++++P < 0.0001 vs. miR-21(−).

Article Snippet: Selected HPASMC monolayers were then placed in a normobaric hypoxia chamber (1% O 2 -5% CO 2 ; Biospherix, Lacuna, NY) or propagated in a cell culture incubator under normoxic conditions (21% O 2 -5% CO 2 ) for 48–72 h. In selected studies, to activate PPARγ, the pharmacological PPARγ agonist RSG (10–25 μM) or an equal volume of dimethyl sulfoxide (DMSO; 1%; Fisher Scientific, Fair Lawn, NJ) vehicle was added to the HPASMC culture media.

Techniques: Expressing, Transfection, Functional Assay, Western Blot, Control, Incubation, Quantitative RT-PCR, Over Expression, Activity Assay, Cell Counting

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Peroxisome proliferator-activated receptor-γ enhances human pulmonary artery smooth muscle cell apoptosis through microRNA-21 and programmed cell death 4

doi: 10.1152/ajplung.00532.2016

Figure Lengend Snippet: PPARγ modulates hypoxia-induced alterations in miR-21 and PDCD4 expression to enhance apoptosis and attenuate HPASMC proliferation. The schema depicts the proposed signaling interactions involved in hypoxia-induced HPASMC proliferation. Hypoxia-induced HPASMC proliferation is associated with enhanced expression of miR-21, which causes posttranscriptional reduction in PDCD4 gene translation and contributes to apoptosis resistance. Activation of PPARγ inhibits HPASMC proliferation by blunting the excitatory effects of hypoxia on miR-21 expression, which derepresses PDCD4 and restores the susceptibility of HPASMCs to undergo apoptosis. Collectively, a finely tuned balance of mediators of proliferation and apoptosis is required to maintain vascular wall cell homeostasis.

Article Snippet: Selected HPASMC monolayers were then placed in a normobaric hypoxia chamber (1% O 2 -5% CO 2 ; Biospherix, Lacuna, NY) or propagated in a cell culture incubator under normoxic conditions (21% O 2 -5% CO 2 ) for 48–72 h. In selected studies, to activate PPARγ, the pharmacological PPARγ agonist RSG (10–25 μM) or an equal volume of dimethyl sulfoxide (DMSO; 1%; Fisher Scientific, Fair Lawn, NJ) vehicle was added to the HPASMC culture media.

Techniques: Expressing, Activation Assay

Journal: The EMBO Journal

Article Title: The nuclear receptor PPAR? individually responds to serotonin- and fatty acid-metabolites

doi: 10.1038/emboj.2010.197

Figure Lengend Snippet: Configurations of indole acetate-containing ligands and known agonists in the PPARγ LBD. (A) Superposition of known agonists in PPARγ LBDs. Full agonists (orange) and partial ones (cyan) are shown within the apo-LBD (2ZK0; Waku et al, 2009a). The Cα atoms of the LBD are coloured yellow (helix H12), red (Ω loop), blue (β-sheet), and grey (other region). Full agonists are from PDB 2PRG (Nolte et al, 1998); 1FM9 (Gampe et al, 2000); 1I7I (Cronet et al, 2001); 1K74 (Xu et al, 2001); 2ATH (Mahindroo et al, 2005); 2I4J (Pochetti et al, 2007); 2Q59 (Bruning et al, 2007); and 3B3K (Montanari et al, 2008). Partial agonists are from 4PRG: Oberfield et al, 1999); 2Q5P, 2Q5S, 2Q6R, and 2Q61(Bruning et al, 2007); and 3D6D (Montanari et al, 2008). (B) Close-up view of the full agoinsts. Red arcs indicate hydrogen bonds between full agonists and Tyr473. (C) Close-up view of the partial agonists. The area enclosed by the black dashed line is the AF-2 pocket. (D) Chemical structures of IDM, 5-HT, HIA, and MIA. The indole ring and the carboxyl group are coloured red and blue, respectively. (E–H) Crystal structures of the PPARγ LBDs in complex with indole acetate-containing ligands. IDM is coloured cyan (E), HIA is green (F), MIA is yellow (G), and 5-HT is magenta (H), in close-up views with the omit 2Fo-Fc map (contoured at 1σ). The LBD and the hydrogen bonds between each molecule and Tyr473 are represented as described in (A) and (B).

Article Snippet: In addition to its physiological functions within the GI tract, plasma 5-HT also regulates the bone mass and cardiac function ( Dempsie and MacLean, 2008 ; Fligny et al, 2008 ; Yadav et al, 2010 ), which are both reported as the pharmacological effects of full PPARγ agonists ( Nissen and Wolski, 2007 ; Grey, 2009 ).

Techniques:

Journal: The EMBO Journal

Article Title: The nuclear receptor PPAR? individually responds to serotonin- and fatty acid-metabolites

doi: 10.1038/emboj.2010.197

Figure Lengend Snippet: Characterization of indole acetate-containing ligands as AF-2-mediated agonists for PPARγ. (A) Effects of each indole acetate-containing ligand on the activity of the Gal4DBD-PPARγLBD. The final concentrations of BRL49653 (BRL) and IDM were 1 and 10 μM, respectively. The concentrations of 5-HT, HIA, and MIA were 100 μM. The RLU (Luc/YFP) is shown with +s.d. (n=4). (B) Effects on the ligand-induced expression of the endogenous PPARγ-target genes AOX and LXRα in PMA-activated macrophage-like THP-1 cells. After activation by PMA, the cells were treated with 1 μM BLR49653 (BRL), 10 μM IDM, 100 μM HIA, and 100 μM MIA, respectively. β-ACTIN and GAPDH were used as references. (C) Effects on 3T3-L1 adipogenesis. After a pre-treatment with 1 μM Dex, 0.5 mM IBMX, and 5 μg/ml insulin, the cells were incubated with 10 μM 15d-PGJ2, 10 μM IDM, or 100 μM MIA, respectively. Oil red O stained cells are shown at the top and are quantified at the bottom by measurement at the OD550 with ±s.d. The statistical comparison of each ligand with a negative control (DMSO) was accomplished using the Mann–Whitney U-test. *P<0.05. n=4. (D) Effect of the Y473F mutation on the protein stability of Gal4DBD-PPARγLBD. The wild type (WT) and the Y473F mutant were transfected into HEK293T cells. After 1 day, the cells were collected and analysed by immunoblotting with an anti-Gal4 DBD antibody. Mock-transfected cells (-) were used as a negative control, and tubulin was used as a reference. (E) Effects of the Y473F mutation on the ligand-induced activity of Gal4DBD-PPARγLBD. Open and closed bars show the ligand-induced activities of the wild type (WT) and the mutant (Y473F), respectively. BRL49653 (BRL), IDM, and MIA were added at final concentrations of 1, 10, and 100 μM, respectively. The RLU (Luc/YFP) is shown with +s.d. (n=4). (F) Effects of the Y473F mutation on the IDM binding to the PPARγ LBD. The direct interactions of IDM with the wild type (WT) and the Y473F mutant PPARγ LBD were quantitatively analysed by ITC, as shown in the left and right panels, respectively.

Article Snippet: In addition to its physiological functions within the GI tract, plasma 5-HT also regulates the bone mass and cardiac function ( Dempsie and MacLean, 2008 ; Fligny et al, 2008 ; Yadav et al, 2010 ), which are both reported as the pharmacological effects of full PPARγ agonists ( Nissen and Wolski, 2007 ; Grey, 2009 ).

Techniques: Activity Assay, Expressing, Activation Assay, Incubation, Staining, Comparison, Negative Control, MANN-WHITNEY, Mutagenesis, Transfection, Western Blot, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: The nuclear receptor PPARγ selectively inhibits Th17 differentiation in a T cell–intrinsic fashion and suppresses CNS autoimmunity

doi: 10.1084/jem.20082771

Figure Lengend Snippet: Control of Th17 differentiation by PPARγ. (a) MOG-EAE was induced in PIO or vehicle-treated wild-type mice ( n = 6 per group, 3 experiments), and the clinical disease score was assessed daily. (b) In a separate experiment, mice were sacrificed at the peak of disease (day 18), CD4 + T cells were isolated from the CNS, restimulated with PMA/ionomycin, and analyzed by flow cytometry gated on CD4 + T cells; representative dot plots and mean results ± SEM from four animals per group are shown; data are from two experiments. (c) CD4 + T cells from the CNS were restimulated with MOG 35-55 -loaded DCs, and numbers of IL-17 and of IFN-γ–producing cells per 3 × 10 4 CD4 + T cells were determined by ELISpot analysis. Graphs denote mean ± SEM of all animals ( n = 6 per group, 2 experiments). (d) Purified CD4 + T cells from CD4-PPARγ KO mice or WT littermates were treated with PIO or the endogenous PPARγ agonist 13s-HODE, and Th17 differentiation was induced by stimulation for 72 h (top row). Alternatively, Th1 differentiation was induced for 72 h (bottom row). Cytokine-producing cells were determined by flow cytometry after PMA/ionomycin restimulation. Only living cells were analyzed by using LIVE/DEAD stain and exclusion of autofluorescence (x axis). Numbers denote mean percentage ± SEM. (e) Th17 differentiation was induced as above and TNF, IL-17A, and IL-22 expression were determined by flow cytometry. Numbers denote mean percentage ± SEM. (f) Th17 differentiation was induced as above; after 72 h expression of IL-17A, IL-17F, IL-21, and IL-23R were measured by quantitative real-time RT-PCR normalized to β-actin levels. (g) Additionally, CCR6-expression was determined by flow cytometry; CCL20-release was assessed by ELISA. (d–g) One out of at least three independent experiments is shown.

Article Snippet: The endogenous PPARγ agonist 13s-HODE (Cayman Chemicals) was used at a concentration of 10 μM.

Techniques: Control, Isolation, Flow Cytometry, Enzyme-linked Immunospot, Purification, Staining, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay