Journal: Journal of medicinal chemistry

Article Title: A novel N-substituted valine derivative with unique PPARγ binding properties and biological activities

doi: 10.1021/acs.jmedchem.0c01555

Figure Lengend Snippet: Effect of 7j on PPARγ phosphorylation. (A) Percentage of in vitro PPARγ Ser273 phosphorylation by CDK5 in the presence of 0.1 μM of 7j or Rosi. (B) Phosphorylation of PPARγ Ser273 in 3T3-L1 adipocyte incubated for 60 minutes with 5 μM of Rosi, 7j or Roscovitine (Rsv) before TNFα stimulation (50 ng/ml; 90 minutes). Two independent experiments are shown. Phosphospecific antibodies were from Rockland or New England Peptide (NEP). Arrow heads indicate phosphorylated PPARγ and PPARγ2. (C) RT-PCR analysis of the expression levels of a selection of genes known to be regulated by CDK5-dependent phosphorylation of PPARγ. Values are means ± SD (n = 5) expressed relative to the mean of control. **p < 0.01, ***p < 0.001 vs. control (one-way ANOVA followed by Dunnett’s post hoc test).

Article Snippet: PPARγ phospho Ser273 antibodies were from Rockland (Limerick, PA, USA) or custom produced by New England Peptide (Gardner, MA, USA).

Techniques: Phospho-proteomics, In Vitro, Incubation, Reverse Transcription Polymerase Chain Reaction, Expressing, Selection, Control

Journal: Animals : an Open Access Journal from MDPI

Article Title: Effect of Actin Alpha Cardiac Muscle 1 on the Proliferation and Differentiation of Bovine Myoblasts and Preadipocytes

doi: 10.3390/ani11123468

Figure Lengend Snippet: RT-qPCR primer sequence.

Article Snippet: The protein samples in the gel were transferred to a polyvinylidene fluoride membrane (PVDF, Merck Millipore, Taufkirchen, Germany), and then sealed with QuickBlock Western blocking solution (Beyotime Biotechnology, Shanghai, China) for 20 min. GAPDH (rabbit anti-GAPDH, 1:10,000 Abcam, Cambridge, UK, NP_001029206.1), ACTC1 (rabbit anti-ACTC1, 1:1000, Invitrogen, CA, USA NP_001029757.1), MYOD1 (mouse anti-MYOD1, 1:1000, Abcam, Cambridge, NT, UK, NP_001035568.2), MYOG (rabbit anti-MYOG, 1:1000, Abcam, Cambridge, NT, UK, NP_001104795.1), MYHC (mouse anti-MYHC, 1:200, Abcam, Cambridge, NT, UK), MRF4 (mouse anti-MYF6, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA, NP_861527.1), MEF2A (rabbit anti-MEF2A, 1:1000, Abcam, Cambridge, NT, UK, NP_001077107.1), FABP4 (rabbit anti-FABP4, 1:1000, Abcam, NT, UK, NP_776739.1), PPARγ (rabbit anti-PPARγ, 1:1000, Boster, Wuhan, China, NP_851367.1), FASN (rabbit anti-FASN, 1:2000, Abcam, Cambridge, NT, UK, NP_777087.1) antibodies were added and incubated overnight at 4 °C.

Techniques: Sequencing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Effect of Actin Alpha Cardiac Muscle 1 on the Proliferation and Differentiation of Bovine Myoblasts and Preadipocytes

doi: 10.3390/ani11123468

Figure Lengend Snippet: Detection of adipogenic marker gene expression at mRNA level after ectopic expression of ACTC1 . ( a – e ) Detection of mRNA expression of ACTC1 , PPARγ , FABP4 , FASN and SCD1 genes on D0, D2, D4, D6 and D8 days by qRT-PCR after ectopic expression ACTC1 . Each experiment was performed in triplicate. Error bars represent s.e.m. * p < 0.05; ** p < 0.01.

Article Snippet: The protein samples in the gel were transferred to a polyvinylidene fluoride membrane (PVDF, Merck Millipore, Taufkirchen, Germany), and then sealed with QuickBlock Western blocking solution (Beyotime Biotechnology, Shanghai, China) for 20 min. GAPDH (rabbit anti-GAPDH, 1:10,000 Abcam, Cambridge, UK, NP_001029206.1), ACTC1 (rabbit anti-ACTC1, 1:1000, Invitrogen, CA, USA NP_001029757.1), MYOD1 (mouse anti-MYOD1, 1:1000, Abcam, Cambridge, NT, UK, NP_001035568.2), MYOG (rabbit anti-MYOG, 1:1000, Abcam, Cambridge, NT, UK, NP_001104795.1), MYHC (mouse anti-MYHC, 1:200, Abcam, Cambridge, NT, UK), MRF4 (mouse anti-MYF6, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA, NP_861527.1), MEF2A (rabbit anti-MEF2A, 1:1000, Abcam, Cambridge, NT, UK, NP_001077107.1), FABP4 (rabbit anti-FABP4, 1:1000, Abcam, NT, UK, NP_776739.1), PPARγ (rabbit anti-PPARγ, 1:1000, Boster, Wuhan, China, NP_851367.1), FASN (rabbit anti-FASN, 1:2000, Abcam, Cambridge, NT, UK, NP_777087.1) antibodies were added and incubated overnight at 4 °C.

Techniques: Marker, Gene Expression, Expressing, Quantitative RT-PCR

Journal: Animals : an Open Access Journal from MDPI

Article Title: Effect of Actin Alpha Cardiac Muscle 1 on the Proliferation and Differentiation of Bovine Myoblasts and Preadipocytes

doi: 10.3390/ani11123468

Figure Lengend Snippet: Detection of adipogenic marker gene expression at protein level after ectopic expression of ACTC1 . ( a ) Detect the proteins expression of ACTC1, PPARγ, FABP4, FASN and SCD1 on D2, D4, D6 and D8 days by Western blot after ectopic expression of ACTC1 . ( b – f ) Use Image J software to measure the gray value of protein bands. Each experiment was performed in triplicate. Error bars represent s.e.m. * p < 0.05; ** p < 0.01. Original western blot figures in .

Article Snippet: The protein samples in the gel were transferred to a polyvinylidene fluoride membrane (PVDF, Merck Millipore, Taufkirchen, Germany), and then sealed with QuickBlock Western blocking solution (Beyotime Biotechnology, Shanghai, China) for 20 min. GAPDH (rabbit anti-GAPDH, 1:10,000 Abcam, Cambridge, UK, NP_001029206.1), ACTC1 (rabbit anti-ACTC1, 1:1000, Invitrogen, CA, USA NP_001029757.1), MYOD1 (mouse anti-MYOD1, 1:1000, Abcam, Cambridge, NT, UK, NP_001035568.2), MYOG (rabbit anti-MYOG, 1:1000, Abcam, Cambridge, NT, UK, NP_001104795.1), MYHC (mouse anti-MYHC, 1:200, Abcam, Cambridge, NT, UK), MRF4 (mouse anti-MYF6, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA, NP_861527.1), MEF2A (rabbit anti-MEF2A, 1:1000, Abcam, Cambridge, NT, UK, NP_001077107.1), FABP4 (rabbit anti-FABP4, 1:1000, Abcam, NT, UK, NP_776739.1), PPARγ (rabbit anti-PPARγ, 1:1000, Boster, Wuhan, China, NP_851367.1), FASN (rabbit anti-FASN, 1:2000, Abcam, Cambridge, NT, UK, NP_777087.1) antibodies were added and incubated overnight at 4 °C.

Techniques: Marker, Gene Expression, Expressing, Western Blot, Software

Journal: ImmunoTargets and Therapy

Article Title: Identification of CRTH2 as a New PPARγ-Target Gene in T Cells Suggested CRTH2 Dependent Conversion of T h 2 Cells as Therapeutic Concept in COVID-19 Infection

doi: 10.2147/ITT.S463601

Figure Lengend Snippet: Regulated Genes in Jurkat T Cells in Response to the Parallel Incubation with the PPARγ Agonist Rosiglitazone and the Irreversible PPARγ Antagonist GW9662

Article Snippet: Intracellular staining for PPARγ expression was established using the FoxP3 staining buffer set as outlined above and a polyclonal rabbit anti-PPARγ antibody labelled with Alexa Fluor750 (#BSS-BS-4590R-A750, Biozol Diagnostica) and the identical clone unconjugated (#BSS-BS-4590R).

Techniques: Incubation, Binding Assay, Sequencing

Journal: ImmunoTargets and Therapy

Article Title: Identification of CRTH2 as a New PPARγ-Target Gene in T Cells Suggested CRTH2 Dependent Conversion of T h 2 Cells as Therapeutic Concept in COVID-19 Infection

doi: 10.2147/ITT.S463601

Figure Lengend Snippet: T cell transcriptome in response to PPARγ activation/inhibition. Jurkat T cells were transduced with an inducible Tet-on vector system to express PPARγ in response to doxycycline treatment. Following selection of positive clones, cells were treated with the PPARγ agonist rosiglitazone [1 µM] (left column) or the combination of rosiglitazone [1 µM] and the irreversible PPARγ antagonist GW9662 [10 µM] (right column) for 24 h. Afterwards cells were harvested, and mRNA isolated as described in Materials and Methods. Following preparation of NGS libraries and sequencing, transcriptome analyses revealed and induction of 55 genes and the downregulation of 1 gene in response to rosiglitazone treatment. After rosiglitazone treatment in combination with GW9662 only 4 genes were induced, and 38 genes were downregulated.

Article Snippet: Intracellular staining for PPARγ expression was established using the FoxP3 staining buffer set as outlined above and a polyclonal rabbit anti-PPARγ antibody labelled with Alexa Fluor750 (#BSS-BS-4590R-A750, Biozol Diagnostica) and the identical clone unconjugated (#BSS-BS-4590R).

Techniques: Activation Assay, Inhibition, Transduction, Plasmid Preparation, Selection, Clone Assay, Isolation, Sequencing

Journal: ImmunoTargets and Therapy

Article Title: Identification of CRTH2 as a New PPARγ-Target Gene in T Cells Suggested CRTH2 Dependent Conversion of T h 2 Cells as Therapeutic Concept in COVID-19 Infection

doi: 10.2147/ITT.S463601

Figure Lengend Snippet: PPARγ expression in CD4 + T cells derived from intensive care COVID-19 patients. (A) Leukocytes were isolated from blood by erythrocyte lysis. CD4 + T cells were stained with an AmCyan-labeled anti-CD4 antibody and intracellular PPARγ was detected following the protocol for FoxP3 staining using an AlexaFluor750-labeled-anti-PPARγ antibody (left panel; HD, healthy donor; C19, COVID-19 patient). As a control, the PPARγ antibody was included unlabeled 1:10 to the labeled antibody to show specific binding (middle panel; HD, healthy donor; C19, COVID-19 patient; s CP, specific competition). An unspecific antibody (anti-FoxP3-APC), used 1:10, was included as well (right panel: HD, healthy donor; C19, COVID-19 patient; us CP, unspecific competition). (B) Quantification of the relative PPARγ expression (mean fluorescence ± SE) of healthy donors (black bar) compared to intensive care COVID-19 patients (white bar). Unlabeled PPARγ antibody competition was included to show PPARγ-binding specificity (grey bar, n = 5). Samples of the patients were drawn on the first or second day during the ICU stay. *p < 0.05.

Article Snippet: Intracellular staining for PPARγ expression was established using the FoxP3 staining buffer set as outlined above and a polyclonal rabbit anti-PPARγ antibody labelled with Alexa Fluor750 (#BSS-BS-4590R-A750, Biozol Diagnostica) and the identical clone unconjugated (#BSS-BS-4590R).

Techniques: Expressing, Derivative Assay, Isolation, Lysis, Staining, Labeling, Control, Binding Assay, Fluorescence

Journal: ImmunoTargets and Therapy

Article Title: Identification of CRTH2 as a New PPARγ-Target Gene in T Cells Suggested CRTH2 Dependent Conversion of T h 2 Cells as Therapeutic Concept in COVID-19 Infection

doi: 10.2147/ITT.S463601

Figure Lengend Snippet: CRTH2 overexpression enhanced 15-deoxy-∆12,14-prostaglandin J 2 (15d-PGJ 2 )-dependent PPARγ activation in HEK293T cells. (A) CRTH2 expression vectors encoding for CRTH2 (grey column), CRTH2-EGFP (white column) or EGFP (black column) were transiently transfected into HEK293T cells in combination with a PPRE-containing PPARγ reporter plasmid and a renilla-firefly luciferase encoding control vector. Transfected cells were treated with 100 nM, 1 µM or 10 µM 15d-PGJ 2 or 10 µM rosiglitazone (PPARγ agonist). PPARγ-dependent transactivation was followed as relative luciferase units (RLU), which is the quotient from PPRE-driven firefly-luciferase activity by the CMV-induced renilla luciferase activity as transfection efficiency control, normalized to the untreated controls (= 1) (n = 6–11). (B) HEK293T cells transiently transfected with a CRTH2 expression vector and the PPARγ reporter system as described in ( A ), were treated with 1 µM 15d-PGJ 2 and 100 nM, 1 µM, or 10 µM of the CRTH2 inhibitor fevipripant or remained untreated as a control. PPARγ transactivation was determined as in ( A ) (n = 4–7). *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Intracellular staining for PPARγ expression was established using the FoxP3 staining buffer set as outlined above and a polyclonal rabbit anti-PPARγ antibody labelled with Alexa Fluor750 (#BSS-BS-4590R-A750, Biozol Diagnostica) and the identical clone unconjugated (#BSS-BS-4590R).

Techniques: Over Expression, Activation Assay, Expressing, Transfection, Plasmid Preparation, Luciferase, Control, Activity Assay