Journal: Frontiers in Pharmacology

Article Title: Menisoxoisoaporphine A, a novel oxoisoaporphine alkaloid from Menispermi Rhizoma, inhibits inflammation by targeting PDE4B

doi: 10.3389/fphar.2024.1505116

Figure Lengend Snippet: Primer sequences used in this study.

Article Snippet: PDE4B-IN-3 (IN-3, purity ≥98%) was purchased from MedChemExpress (Shanghai, China).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Menisoxoisoaporphine A, a novel oxoisoaporphine alkaloid from Menispermi Rhizoma, inhibits inflammation by targeting PDE4B

doi: 10.3389/fphar.2024.1505116

Figure Lengend Snippet: siRNA sequences used in the study.

Article Snippet: PDE4B-IN-3 (IN-3, purity ≥98%) was purchased from MedChemExpress (Shanghai, China).

Techniques: Sequencing, Control

Journal: Frontiers in Pharmacology

Article Title: Menisoxoisoaporphine A, a novel oxoisoaporphine alkaloid from Menispermi Rhizoma, inhibits inflammation by targeting PDE4B

doi: 10.3389/fphar.2024.1505116

Figure Lengend Snippet: RNA-seq analysis revealed the anti-inflammatory properties of Menisoxoisoaporphine A (MA) were associated with PDE4B. RAW264.7 cells were stimulated with 1 μg·mL -1 lipopolysaccharide (LPS), and concurrently treated with MA (0, 12 μM) for 12 h. The cells were harvested for transcriptome profiling by RNA-seq. (A) Volcano plot showing DEGs between control vs . model groups. (B) Volcano plot showing DEGs between model vs . MA groups. (C) Venn diagram showing the co-differentially expressed genes (co-DEGs) between control vs . model and model vs . MA groups. (D) Heatmap showing the expression of 117 co-DEGs. (E) The mRNA relative expression of Pde4b, Igf2r, Slc6a12, Itgb7, Tlr9, Nox1, Mmp12, Abca1, Cd80, Il11 and Tnfrsf9 were validated by qRT-PCR in RAW264.7 cells. For the volcano plot, the abscissa represents the Log2 transformed fold-change, and the ordinate represents the -log10 transformed p -value. The green dots indicated the downregulated genes, the red dots indicated the upregulated genes and the grey dots indicated the non-DEGs. DEGs: differentially expressed genes (FPKM value, fold change ≥1.5 and p ≤ 0.05). Control (C1-C3): control normal group; Model (M1-M3): only LPS-treated group; MA (MA1-MA3): LPS and MA-treated group. Each value was expressed as the means ± SEM (n = 3). # p < 0.05: vs . control group. * p < 0.05: vs . only LPS-treated group.

Article Snippet: PDE4B-IN-3 (IN-3, purity ≥98%) was purchased from MedChemExpress (Shanghai, China).

Techniques: RNA Sequencing Assay, Control, Expressing, Quantitative RT-PCR, Transformation Assay

Journal: Frontiers in Pharmacology

Article Title: Menisoxoisoaporphine A, a novel oxoisoaporphine alkaloid from Menispermi Rhizoma, inhibits inflammation by targeting PDE4B

doi: 10.3389/fphar.2024.1505116

Figure Lengend Snippet: Menisoxoisoaporphine A (MA) inhibited LPS-induced inflammation via PDE4B-cAMP-PKA-NF-κB pathway. RAW264.7 cells were stimulated with 1 μg·mL -1 lipopolysaccharide (LPS), and concurrently treated with MA (0, 12 μM) or PDE4B-IN-3 (IN-3, 1 μM) for 12 h. (A) Representative Western blot bands and (B–F) quantification of PDE4B, cAMP, p-PKA, p-P65(Ser276)/P65 and p-IκB(Ser32/36)/IκB. β-Actin was used as an internal reference. Each value was expressed as the means ± SEM (n = 3). # p < 0.05: vs . control group. * p < 0.05: vs . only LPS-treated group.

Article Snippet: PDE4B-IN-3 (IN-3, purity ≥98%) was purchased from MedChemExpress (Shanghai, China).

Techniques: Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Menisoxoisoaporphine A, a novel oxoisoaporphine alkaloid from Menispermi Rhizoma, inhibits inflammation by targeting PDE4B

doi: 10.3389/fphar.2024.1505116

Figure Lengend Snippet: PDE4B knockdown abolished the anti-inflammatory effects of Menisoxoisoaporphine A (MA) at LPS-induced RAW264.7 cells. RAW264.7 cells were transfected with Pde4b siRNA or control siRNA for 12 h. (A) The PDE4B proteins were probed by western bolt analysis. Transfected cells were then stimulated with 1 μg·mL −1 lipopolysaccharide (LPS), and concurrently treated with MA (0, 12 μM) for 12 h. (B) Representative Western blot banding pictures and quantification of PDE4B, cAMP, p-PKA, p-IκB(Ser32/36)/IκB and p-P65(Ser276)/P65 of Pde4b siRNA#2 treatment. (C) Representative Western blot banding pictures and quantification of PDE4B, cAMP, p-PKA, p-IκB(Ser32/36)/IκB and p-P65(Ser276)/P65 of Pde4b siRNA#4 treatment. β-Actin was used as an internal reference. NC, cells without any treatment. SC, cells only treated with control siRNA. Each value was expressed as the means ± SEM (n = 3). # p < 0.05: vs . control siRNA group. * p < 0.05: vs . indicated group.

Article Snippet: PDE4B-IN-3 (IN-3, purity ≥98%) was purchased from MedChemExpress (Shanghai, China).

Techniques: Knockdown, Transfection, Control, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Menisoxoisoaporphine A, a novel oxoisoaporphine alkaloid from Menispermi Rhizoma, inhibits inflammation by targeting PDE4B

doi: 10.3389/fphar.2024.1505116

Figure Lengend Snippet: Menisoxoisoaporphine A (MA) inhibited cAMP/PKA-NF-κB via PDE4B. RAW264.7 cells were transfected with pIRES2-EGFP-Pde4b overexpressed plasmid or empty pIRES2-EGFP for 12 h. (A) PDE4B protein expression detected by western bolt. Transfected cells were then stimulated with 1 μg·mL −1 lipopolysaccharide (LPS), and concurrently treated with MA (0, 12 μM) for 12 h. (B) Representative Western blot bands and quantification of PDE4B, cAMP, p-PKA, p-P65(Ser276)/P65 and p-IκB(Ser32/36)/IκB. β-Actin was used as an internal reference. Each value was expressed as the means ± SEM (n = 3). # p < 0.05: vs . control hGFP group. * p < 0.05: vs . indicated group.

Article Snippet: PDE4B-IN-3 (IN-3, purity ≥98%) was purchased from MedChemExpress (Shanghai, China).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Menisoxoisoaporphine A, a novel oxoisoaporphine alkaloid from Menispermi Rhizoma, inhibits inflammation by targeting PDE4B

doi: 10.3389/fphar.2024.1505116

Figure Lengend Snippet: Menisoxoisoaporphine A (MA) could directly bind to PDE4B at Tyr405 site. (A) Molecular docking simulation between MA and PDE4B. (B) The root mean square deviation (RMSD) of PDE4B in the PDE4B WT system and PDE4B Y405A system. (C) The RMSD of MA in the PDE4B WT system and PDE4B Y405A system. (D) The residual energy decomposition of the binding free energy of PDE4B WT system and PDE4B Y405A system, the horizontal axis denotes the respective amino acids associated with each position. WT: wild type; TYR: tyrosine; Y405A: The tyrosine residue at position 405 is mutated to glycine.

Article Snippet: PDE4B-IN-3 (IN-3, purity ≥98%) was purchased from MedChemExpress (Shanghai, China).

Techniques: Binding Assay, Residue

Journal: Frontiers in Pharmacology

Article Title: Menisoxoisoaporphine A, a novel oxoisoaporphine alkaloid from Menispermi Rhizoma, inhibits inflammation by targeting PDE4B

doi: 10.3389/fphar.2024.1505116

Figure Lengend Snippet: Binding free energy and different energy contributions of MA-PDE4B WT systems and MA-PDE4B Y405A systems (kcal/mol).

Article Snippet: PDE4B-IN-3 (IN-3, purity ≥98%) was purchased from MedChemExpress (Shanghai, China).

Techniques: Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Menisoxoisoaporphine A, a novel oxoisoaporphine alkaloid from Menispermi Rhizoma, inhibits inflammation by targeting PDE4B

doi: 10.3389/fphar.2024.1505116

Figure Lengend Snippet: Menisoxoisoaporphine A (MA) inhibited cAMP-PKA-NF-κB mediated inflammation via targeting PDE4B.

Article Snippet: PDE4B-IN-3 (IN-3, purity ≥98%) was purchased from MedChemExpress (Shanghai, China).

Techniques:

Journal: iScience

Article Title: Using brain cell-type-specific protein interactomes to interpret neurodevelopmental genetic signals in schizophrenia

doi: 10.1016/j.isci.2023.106701

Figure Lengend Snippet:

Article Snippet: PDE4B , Cell Signaling Technology , Cat# 72096S; RRID: AB_2799812.

Techniques: Control, Recombinant, Sterility, Magnetic Beads, Lysis, Software

Journal: Antioxidants

Article Title: Rolipram Prevents the Formation of Abdominal Aortic Aneurysm (AAA) in Mice: PDE4B as a Target in AAA

doi: 10.3390/antiox10030460

Figure Lengend Snippet: PDE4B expression is up-regulated in the abdominal aortas from AngII-infused ApoE −/− mice. ApoE −/− mice were infused with AngII (1000 ng/kg/min) or saline solution for 28 days. ( A ) The PDE4B expression in abdominal aortas from these animals was assessed by Western blot. The levels of β-actin are shown as a loading control. The boxplot on the right shows the quantification of the PDE4B protein levels. The box extends from the 25th to the 75th percentile, and the median is indicated by a horizontal line. The whiskers represent the maximum and minimum values (Saline, n = 4; Ang II n = 6); * p < 0.01 vs. saline. ( B ) Representative PDE4B immunostaining in these samples. The arrowheads indicate the PD4B-positive cells in aneurysmal tissues. Bars: 20 µm.

Article Snippet: Then, the samples were incubated overnight at 4 °C with the following primary antibodies: MAC3 (sc-19991, Santa Cruz Biotechnology, Dallas, TX, USA), CD3 (A0452, Dako, Agilent Technologies), neutrophil elastase (M0752, Dako, Agilent Technologies), MCP1 (sc-1785, Santa Cruz Biotechnology), or PDE4B (NBP2-01171, Novus Biologicals).

Techniques: Expressing, Saline, Western Blot, Control, Immunostaining

Journal: Antioxidants

Article Title: Rolipram Prevents the Formation of Abdominal Aortic Aneurysm (AAA) in Mice: PDE4B as a Target in AAA

doi: 10.3390/antiox10030460

Figure Lengend Snippet: PDE4B expression is enhanced in human AAA. ( A ) PDE4B expression in abdominal aorta from AAA patients (AAA) and healthy donors (Do), assessed by real-time PCR. The data are presented as boxplots. The box extends from the 25th to the 75th percentile, and the median is indicated by a horizontal line. The whiskers represent the maximum and minimum values (DO, n = 14; AAA n = 61); * p < 0.001 vs. Donors. ( B ) Representative PDE4B immunostaining in healthy aorta from donors and aneurysmal tissues from AAA patients (top panels; bar: 100 µm). The boxed areas are shown at a higher magnification below, and the arrowheads indicate PDE4B-positive cells (lower panels; bar: 50 µm). ( C ) PDE4D mRNA levels in these samples.

Article Snippet: Then, the samples were incubated overnight at 4 °C with the following primary antibodies: MAC3 (sc-19991, Santa Cruz Biotechnology, Dallas, TX, USA), CD3 (A0452, Dako, Agilent Technologies), neutrophil elastase (M0752, Dako, Agilent Technologies), MCP1 (sc-1785, Santa Cruz Biotechnology), or PDE4B (NBP2-01171, Novus Biologicals).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunostaining

Journal: Nature Communications

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

doi: 10.1038/ncomms10856

Figure Lengend Snippet: PDE4B was cloned from mouse hepatocyte cDNA. The recombinant protein was overexpressed in E. coli and purified. PDE protein was phosphorylated for 1 h with purified recombinant activated AMPK and/or purified PKA catalytic subunits and [γ- 32 P] ATP, and analysed by SDS–PAGE followed by Coomassie blue staining and phosphorimaging for quantification ( a , c ). In b , PDE was phosphorylated for 1 h with recombinant activated AMPK and [γ- 32 P]. Phosphorylation sites were identified by LC–MS/MS after trypsin digestion and radioactive peak separation by high-performance liquid chromatography (HPLC). The phosphorylation sites that were identified are underlined in the right hand panel. In d and e , recombinant PDE was phosphorylated as above but with non-radioactive ATP for PDE assay as indicated. In d , separate determinations of V max and K M were made by linear regression of double reciprocal (Lineweaver Burk) plots. In e , the basal PDE activities of the wild-type (WT), S118A, S125A and S304A mutant proteins were 1.97±0.25, 0.14±0.01, 1.59±0.15 and 0.32±0.09 mU per mg of protein, respectively. Values are means±s.e.m. for n =3 ( c – e ) separate experiments. Statistical analysis was by a paired Student's t -test. *Indicates a significant difference ( P <0.05) compared with control incubations or between the indicated conditions.

Article Snippet: Anti-total ACC (Merck Millipore, Catalogue No. 04-322), anti-P-Ser79-ACC (Merck-Millipore, Catalogue No. 07-303), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Merck-Millipore, Catalogue No. MAB374), anti-total GP (Sigma, Catalogue No. HPA000962), anti-total AMPK β1(R&D Systems, Catalogue No. AF2854) and anti-total AMPK β2 (R&D Systems, Catalogue No. MAB3808), anti-PThr172-AMPK (T172) (Cell Signaling Technologies, Catalogue No. 2535, anti-P-AMPK-substrate (Cell Signaling Technologies, Catalogue No. 5759), anti-P-PKA-substrate (Cell Signaling Technologies, Catalogue No. 9624), anti-total Raptor (Cell Signaling Technologies, Catalogue No. 2280) and anti-P-Ser792-Raptor (Cell Signaling Technologies, Catalogue No. 2083), anti-total CREB (Cell Signaling Technologies, Catalogue No. 9197), anti-phospho-Ser133-CREB (Cell Signaling Technologies, Catalogue No. 9198) and anti-total PDE4B (Origene, Catalogue No. TA503471) antibodies were from the sources cited.

Techniques: Clone Assay, Recombinant, Purification, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, High Performance Liquid Chromatography, Mutagenesis

Journal: Nature Communications

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

doi: 10.1038/ncomms10856

Figure Lengend Snippet: In a , wild-type (WT) or mutant recombinant mouse liver PDE4B was incubated for 1 h with non-radioactive ATP in the presence (+) or absence (−) of recombinant activated AMPK. Proteins (0.1 μg) were seperated by SDS–PAGE for immunoblotting with the indicated antibodies. In b and c , mouse hepatocytes from either WT ( b ) or both WT and AMPK α 1 −/− α 2 LS−/− mice ( c ) were serum-starved overnight and incubated for 1 h with the indicated concentrations of 991 or phenformin. The cells were collected and lysed for immunoblotting with the indicated antibodies, except for PDE4B, which was immunoprecipitated as described in the Methods section, before immunoblotting. In c , phosphorylation levels of AMPK and its targets ACC, Raptor and PDE4B were quantified by densitometry and expressed relative to the corresponding total protein levels or GAPDH before normalization as indicated. Representative immunoblots are shown and for blot quantification in c , the values are means±s.e.m. for n =3 (p-ACC, p-Raptor and p-AMPK) or n =4 (p-PDE4B) separate experiments. Statistical analysis was by a paired Student's t -test. *Indicates a significant difference ( P <0.05).

Article Snippet: Anti-total ACC (Merck Millipore, Catalogue No. 04-322), anti-P-Ser79-ACC (Merck-Millipore, Catalogue No. 07-303), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Merck-Millipore, Catalogue No. MAB374), anti-total GP (Sigma, Catalogue No. HPA000962), anti-total AMPK β1(R&D Systems, Catalogue No. AF2854) and anti-total AMPK β2 (R&D Systems, Catalogue No. MAB3808), anti-PThr172-AMPK (T172) (Cell Signaling Technologies, Catalogue No. 2535, anti-P-AMPK-substrate (Cell Signaling Technologies, Catalogue No. 5759), anti-P-PKA-substrate (Cell Signaling Technologies, Catalogue No. 9624), anti-total Raptor (Cell Signaling Technologies, Catalogue No. 2280) and anti-P-Ser792-Raptor (Cell Signaling Technologies, Catalogue No. 2083), anti-total CREB (Cell Signaling Technologies, Catalogue No. 9197), anti-phospho-Ser133-CREB (Cell Signaling Technologies, Catalogue No. 9198) and anti-total PDE4B (Origene, Catalogue No. TA503471) antibodies were from the sources cited.

Techniques: Mutagenesis, Recombinant, Incubation, SDS Page, Western Blot, Immunoprecipitation

Journal: Nature Communications

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

doi: 10.1038/ncomms10856

Figure Lengend Snippet: Unlike biguanides, treatment with 991 activates AMPK without increasing cellular AMP levels. Both biguanides and 991 activate the major PDE 4B isoenzyme in hepatocytes in an AMPK-dependent manner. Metformin and phenformin activate hepatic AMPK either directly or via a rise in AMP, which could compete with ATP to inhibit adenylate cyclase. Phosphorylation-induced activation of PDE4B by AMPK reduces glucagon-stimulated cAMP accumulation. As a consequence, PKA activation by glucagon and downstream signalling are decreased in hepatocytes incubated with 991, the effect being AMPK-dependent.

Article Snippet: Anti-total ACC (Merck Millipore, Catalogue No. 04-322), anti-P-Ser79-ACC (Merck-Millipore, Catalogue No. 07-303), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Merck-Millipore, Catalogue No. MAB374), anti-total GP (Sigma, Catalogue No. HPA000962), anti-total AMPK β1(R&D Systems, Catalogue No. AF2854) and anti-total AMPK β2 (R&D Systems, Catalogue No. MAB3808), anti-PThr172-AMPK (T172) (Cell Signaling Technologies, Catalogue No. 2535, anti-P-AMPK-substrate (Cell Signaling Technologies, Catalogue No. 5759), anti-P-PKA-substrate (Cell Signaling Technologies, Catalogue No. 9624), anti-total Raptor (Cell Signaling Technologies, Catalogue No. 2280) and anti-P-Ser792-Raptor (Cell Signaling Technologies, Catalogue No. 2083), anti-total CREB (Cell Signaling Technologies, Catalogue No. 9197), anti-phospho-Ser133-CREB (Cell Signaling Technologies, Catalogue No. 9198) and anti-total PDE4B (Origene, Catalogue No. TA503471) antibodies were from the sources cited.

Techniques: Activation Assay, Incubation