intracellular camp levels  (R&D Systems)

Journal: Nature Communications

Article Title: Integrative residue-intuitive machine learning and MD Approach to Unveil Allosteric Site and Mechanism for β2AR

doi: 10.1038/s41467-024-52399-y

Figure Lengend Snippet: Blue-shaded area: the orthosteric site. Green-shaded area: the allosteric site. Pink-shaded area: the intracellular activation region. a The shortest pathways with the highest frequency from the allosteric site to the orthosteric site in the β2AR-NE-ZINC5042 and β2AR-ALE-ZINC5042 systems. b The shortest pathway with the highest frequency from the orthosteric site to the intracellular activation region. PSN was calculated based on the last 1000-ns trajectory of three independent 1.5-μs cMD simulations for each system.

Article Snippet: To examine the intracellular cAMP levels of HEK293 cells overexpressing β2AR in response to the two agonists (NE (TargetMol, T7044), ALE (Med Chem Express (MCE), HY-B0447B)) under study and allosteric ligand screened (ZINC5042(MCE, HY-108999A), ZINC252008995(MCE, HY-15337), ZINC4213962(MCE, HY-100572), ZINC11681534 (MCE, HY-B0203A)).

Techniques: Activation Assay

Journal: Bioactive Materials

Article Title: Screening of a quinonoid compounds library identifies decylubiquinone as an antioxidant and anti-apoptotic agent against glucocorticoid-induced osteoporosis via CD39/CD73/adenosine axis

doi: 10.1016/j.bioactmat.2026.03.062

Figure Lengend Snippet: Screening of the quinonoid compounds for the treatment of GIOP. (A) Flowchart depicting the screening process of the quinonoid compounds library. The schematic diagram was created by using BioRender.com. (B) Volcano diagram showing the effects of the 153 quinonoid compounds on Runx2 expression in BMSCs. Red and blue dots indicate the specific compounds that up- and down-regulate Runx2 expression in BMSCs, respectively. (C) Heat map showing the effect of the compounds on ALP activity in primary BMSCs. Color from blue to red indicates the ALP activity in primary BMSCs from low to high. (D) Measurement of intracellular ROS level in primary BMSCs treated with three potential compounds by using the fluorescent dye DCFDA. (E) Chemical structure of DUB, the final candidate among the screened drugs. (F) MTT assay for the proliferation of BMSCs treated with different doses of DUB for 2 and 10 days, under osteogenic induction conditions with or without 10 μM Dex. (G) Representative images and quantitative analysis of mineralized nodule formation via Alizarin Red S (ARS) staining in primary BMSCs treated with DUB at a series of concentrations, under osteogenic induction conditions with or without 10 μM Dex. (H) Western blot and quantification for the expression of osteogenesis-related proteins in primary BMSCs under different treatments. (I) Oil Red O staining and quantifications for lipid droplets in primary BMSCs of different groups. n = 4 independent repeats by using different biological samples in each group for in vitro experiments. Data were means ± s.e.m. ∗∗∗ p < 0.001 by one-way ANOVA. Scale bars: 200 μm (G), and 50 μm (I).

Article Snippet: The intracellular cAMP level was examined by using a cAMP ELISA Kit (E-EL-0056, Elabscience, Wuhan, China) according to the manufacturer's instructions.

Techniques: Expressing, Activity Assay, MTT Assay, Staining, Western Blot, In Vitro

Journal: Bioactive Materials

Article Title: Screening of a quinonoid compounds library identifies decylubiquinone as an antioxidant and anti-apoptotic agent against glucocorticoid-induced osteoporosis via CD39/CD73/adenosine axis

doi: 10.1016/j.bioactmat.2026.03.062

Figure Lengend Snippet: Roles of A 2b R in ADO-mediated activation of the cAMP/PKA/CREB pathway in primary BMSCs. ( A ) Principal component analysis (PCA) of RNA-seq data from primary BMSCs treated with Dex or Dex + ADO. ( B ) The volcano plot presented the differentially expressed genes (DEGs) as determined by RNA-Seq in primary BMSCs treated with Dex or Dex + ADO. ( C ) Gene Ontology (GO) enrichment analysis in the biological process category for DEGs as determined by RNA-Seq in primary BMSCs treated with Dex, or Dex + ADO. ( D ) The molecular docking of ADO with mus musculus A 1 R, A 2a R, A 2b R, and A 3 R proteins. ADO is displayed in Cyan. The surrounding residues in the binding pocket are shown in green (forming a non-hydrogen bond with ADO) or magenta (forming a hydrogen bond with ADO). The hydrogen bond is labeled as yellow dashed lines. The backbone of the receptor is depicted as gray. ( E ) RT-qPCR analysis of the mRNA levels of Adora1 , Adora2a , Adora2b , and Adora3 in primary BMSCs treated with vehicle, Dex, or Dex + ADO. ( F ) RT-qPCR analysis for the expression of Runx2 in primary BMSCs of different groups. (G) Gene Set Enrichment Analysis (GSEA) plot showing the differentially expressed pathway (cAMP) between the Dex group and the Dex + ADO group as indicated by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. ( H ) Western blot validation for the knockdown deficiency of A 2b R after transfection with si Adora2b . ( I ) ELISA analysis for the relative intracellular cAMP levels in BMSCs of different groups. ( J ) Western blot and quantification for the expression of PKA, p-PKA, CREB, and p-CREB in primary BMSCs. ( K ) Representative images and quantitative analysis of Alizarin Red S staining for mineralization deposit in primary BMSCs of different groups under osteogenic conditions. n = 4 independent repeats by using different biological samples in each group for in vitro experiments. Data were means ± s.e.m. ns p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 by one-way ANOVA. Scale bar: 200 μm (K).

Article Snippet: The intracellular cAMP level was examined by using a cAMP ELISA Kit (E-EL-0056, Elabscience, Wuhan, China) according to the manufacturer's instructions.

Techniques: Activation Assay, RNA Sequencing, Binding Assay, Labeling, Quantitative RT-PCR, Expressing, Western Blot, Biomarker Discovery, Knockdown, Transfection, Enzyme-linked Immunosorbent Assay, Staining, In Vitro

Journal: Cell Reports Medicine

Article Title: Tumor-specific but immunosuppressive CD39 + CD8 + T cells exhibit double-faceted roles in clear cell renal cell carcinoma

doi: 10.1016/j.xcrm.2025.102360

Figure Lengend Snippet: Tumor-infiltrating CD39 + CD8 + T cells exhibit enhanced cAMP and TCR signaling (A and B) The ordering of CD8 + T cells with clonotypes that overlap between peripheral blood and tumor along pseudotime. (A) Annotated with the pseudotime ordering (left) and sample origin (right). (B) Annotated with CD8 + T cell subpopulations classified by PD-1 and CD39 expression. (C) Relative expression of selected genes related to T cell differentiation, according to the pseudotime. (D) Relative CREM expression according to the CD39 ADT expression. Trend line and error are for linear regression with 95% CI. Pearson’s R 2 and two-sided p value are listed. (E) Table showing the number of cells for six tumor-specific T cell clones shared between blood and tumor (top). Clone #4 was the most abundant in blood ( n = 233) and was also detected at relatively high frequency in tumor ( n = 24). Distribution of clone #4 cells along the pseudotime trajectory (bottom). (F) Volcano plot showing differential gene expression between tumor-specific CD8 + T cells of clone #4 from blood and tumor. (G) Relative concentration of intracellular cAMP within CD39 – and CD39 + CD8 + TILs ( n = 8). Fold change values are shown relative to CD39 − CD8 + TILs within each sample. ∗∗ p < 0.01 by Wilcoxon matched-pairs signed-rank test. (H) CD39 expression in naive CD8 + T cells from PBMCs following anti-CD3 stimulation under indicated conditions: control, RP-cAMPS (cAMP-reducing agent), forskolin (cAMP-elevating agent), and forskolin + RP-cAMPS. Representative flow cytometry plots are shown on the left. The graph on the right shows the proportion of CD39 + cells under each condition, with data points from the same donor connected by lines. Pairwise Wilcoxon signed-rank tests with Benjamini-Hochberg correction for multiple comparisons. ∗∗ p < 0.01, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Intracellular cAMP levels were modulated by the addition of Forskolin (10 μM; Tocris) and/or cAMPS-Rp (1 mM; Tocris).

Techniques: Expressing, Cell Differentiation, Clone Assay, Gene Expression, Concentration Assay, Control, Flow Cytometry