pde inhibitor ibmx  (Nacalai)

Journal: bioRxiv

Article Title: Distinct cAMP regulation in scleroderma lung and skin myofibroblasts governs their dedifferentiation via p38α inhibition

doi: 10.1101/2025.02.26.640163

Figure Lengend Snippet: ( A ) Illustration depicting the AC activating effects of the Gαs-coupled GPCR agonists butaprost, PGE 2 and CAY10684 at EP2 and/or EP4 resulting in an increase in cAMP levels. PGE 2 exhibits inhibitory actions on AC by its ligation of Gαi-coupled EP3, while the EP3 antagonist L-798,106 prevents such AC inhibition. ( B ) qPCR data representing the relative gene expression of the PTGER2 , PTGER3 and PTGER4 genes encoding EP2, EP3 and EP4, respectively, in SSc lung and skin MFs utilized in our experiments. Each symbol represents an individual patient-derived cell line. ( C-E ) Dose response curves of SSc lung and skin MFs treated for 2 h with PGE 2 (designated EP1-4 agonist) ( C ), butaprost ( D ) or CAY 10684 ( E ), measured by cAMP-GLO assay; results are expressed relative to effects of forskolin as maximal stimulus. cAMP quantification in C-E is expressed as a proportion of maximum forskolin-mediated cAMP generation and was performed in the presence of the pan-PDE inhibitor IBMX (500 µM). ( F-H ) Intracellular cAMP concentration (pmol/mL) measured via ELISA in SSc lung and skin MFs following five-minute treatment with PGE 2 (1 µM) ( F ), butaprost (1 µM) ( G ) or CAY10684 (1 µM) ( H ). Data points represent distinct patient-derived cell lines. Significance for comparisons in B was determined by 2-tailed unpaired or paired t-test where appropriate. Significance for cAMP data in C-E ( n = 5) and F-H ( n = 4-6) was determined by two-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.

Article Snippet: PGE 2 (1 μM); the EP2 agonist butaprost (1 μM); the EP4 agonist CAY10684 (1 μM); the EP3 antagonist L-798, 106 (250 nM); the PDE4 inhibitor Ro 20-1724 (100 μM); and the pan-PDE inhibitor IBMX (500 μM) were purchased from Cayman Chemicals (14010, 13740, 15966, 11129, 18272, 13347).

Techniques: Ligation, Inhibition, Gene Expression, Derivative Assay, Glo Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: ANP and BNP Exert Anti-Inflammatory Action via NPR-1/cGMP Axis by Interfering with Canonical, Non-Canonical, and Alternative Routes of Inflammasome Activation in Human THP1 Cells

doi: 10.3390/ijms22010024

Figure Lengend Snippet: cGMP but not PKG-I is involved in non-canonical and alternative inflammasome activation. THP-1 cells were pre-treated for 10 min with ANP (10 −8 M) or BNP (10 −8 M) or cGMP (100 µM 8-Br-cGMP) in the absence or presence of LPS (10 µg/mL for 20 min) + ATP (5 mM for 40 min). When KT-5823 (1 μM) or 3-isobutyl-1-methylxanthine (IBMX) (10 μM) were used, they were added 1 h before ANP, BNP, or 8-Br-cGMP treatment. Cell lysates were immunoblotted for active Cathepsin B ( A ), Gasdermin D ( B , E ), RIPK1 ( C ), or Caspase-8 ( D , F ). Protein loading was assessed by re-probing the blots with an anti-β-actin antibody. One representative image is shown. Representative western blots images are shown. Histograms of densitometric quantification. Bars represent the ratio between respective protein and β-actin band intensity. Untreated cells were used as control and assumed as 1. All histograms indicate mean ± SD of at least n = three independent experiments, each one tested in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 versus untreated cells; ° p < 0.05, °° p < 0.01 and °°° p < 0.001 versus LPS + ATP treated cells; # p < 0.05 versus KT-5823 + LPS + ATP treated cells.

Article Snippet: Human BNP was obtained from Phoenix Europe GmbH and dissolved in H 2 O. LPS (from Escherichia coli 0111: B4), 8-Bromoguanosine 3′,5′-cyclic monophosphate sodium salt (8-Br-cGMP), ATP (dissolved in H 2 O), and the PKG inhibitor KT-5823 and the PDEs inhibitor IBMX (3-isobutyl-1-methylxanthine) (dissolved in 100 % DMSO), were purchased from Merck KGaA (Darmstadt, Germany).

Techniques: Activation Assay, Western Blot, Control