Journal: International Journal of Molecular Sciences

Article Title: Anti-Inflammatory Effects of miR-369-3p via PDE4B in Intestinal Inflammatory Response

doi: 10.3390/ijms25158463

Figure Lengend Snippet: In silico analysis of predicted target interaction between miR-369-3p and Pde4b mRNA. ( A ) miR-369-3p regulates Pde4b mRNA expression, aligning with the 3′UTR region through two binding sites. ( B ) The complementary regions highlighted in white of miR-369-3p and Pde4b 3′UTR are well conserved among the species.

Article Snippet: The primary antibodies used were rabbit mAb anti-PDE4B (ab170939, Abcam, Cambridge, UK, dilution 1:1000), rabbit mAb anti-pCREB (#9198, Cell Signaling, Technology, Danvers, MA, USA, dilution 1:1000), rabbit mAb anti-CREB (#9197, Cell Signaling, Technology, Danvers, MA, USA, dilution 1:1000), rabbit polyAb anti-PKA C-α (#4782, Cell Signaling, Technology, Danvers, MA, USA, dilution 1:1000), mouse mAb anti-GAPDH (sc-365062, Santa Cruz Biotechnology, Inc., Heidelberg, Germany, dilution 1:1000), and secondary antibodies goat anti-mouse IgG-(H+L)-HRP conjugate (170-6516, Biorad Laboratories, Hercules, CA, USA, dilution 1:500) and goat anti-rabbit IgG-(H+L)-HRP conjugate (31466, Invitrogen, Carlsbad, CA, USA, dilution 1:2500).

Techniques: In Silico, Expressing, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Anti-Inflammatory Effects of miR-369-3p via PDE4B in Intestinal Inflammatory Response

doi: 10.3390/ijms25158463

Figure Lengend Snippet: Modulation of PDE4B expression by miR-369-3p. ( A ) The mRNA expression levels of Pde4b evaluated by qRT-PCR in Raw264.7 cells transfected with 30 and 50 nM of miR-369-3p mimic before and after LPS stimulation. ( B ) Western blot analysis of PDE4B protein expression after miR-369-3p mimic transfection in unstimulated cells and following LPS stimulation. GAPDH was used as a housekeeping protein to normalize the data. Data are representative of four independent experiments. The histograms correspond to mean ± SEM (* p < 0.01; ** p < 0.001; *** p < 0.0001).

Article Snippet: The primary antibodies used were rabbit mAb anti-PDE4B (ab170939, Abcam, Cambridge, UK, dilution 1:1000), rabbit mAb anti-pCREB (#9198, Cell Signaling, Technology, Danvers, MA, USA, dilution 1:1000), rabbit mAb anti-CREB (#9197, Cell Signaling, Technology, Danvers, MA, USA, dilution 1:1000), rabbit polyAb anti-PKA C-α (#4782, Cell Signaling, Technology, Danvers, MA, USA, dilution 1:1000), mouse mAb anti-GAPDH (sc-365062, Santa Cruz Biotechnology, Inc., Heidelberg, Germany, dilution 1:1000), and secondary antibodies goat anti-mouse IgG-(H+L)-HRP conjugate (170-6516, Biorad Laboratories, Hercules, CA, USA, dilution 1:500) and goat anti-rabbit IgG-(H+L)-HRP conjugate (31466, Invitrogen, Carlsbad, CA, USA, dilution 1:2500).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Anti-Inflammatory Effects of miR-369-3p via PDE4B in Intestinal Inflammatory Response

doi: 10.3390/ijms25158463

Figure Lengend Snippet: Immunofluorescence staining of PDE4B in Raw264.7 cells after miR-369-3p mimic transfection. In accordance with Western blot results, representative images showed that miR-369-3p modulated the expression of PDE4B compared to the mock control in both unstimulated and LPS-stimulated conditions. For each sample, three images were captured in different positions. Original magnification, 20×. Scale bar, 50 μm.

Article Snippet: The primary antibodies used were rabbit mAb anti-PDE4B (ab170939, Abcam, Cambridge, UK, dilution 1:1000), rabbit mAb anti-pCREB (#9198, Cell Signaling, Technology, Danvers, MA, USA, dilution 1:1000), rabbit mAb anti-CREB (#9197, Cell Signaling, Technology, Danvers, MA, USA, dilution 1:1000), rabbit polyAb anti-PKA C-α (#4782, Cell Signaling, Technology, Danvers, MA, USA, dilution 1:1000), mouse mAb anti-GAPDH (sc-365062, Santa Cruz Biotechnology, Inc., Heidelberg, Germany, dilution 1:1000), and secondary antibodies goat anti-mouse IgG-(H+L)-HRP conjugate (170-6516, Biorad Laboratories, Hercules, CA, USA, dilution 1:500) and goat anti-rabbit IgG-(H+L)-HRP conjugate (31466, Invitrogen, Carlsbad, CA, USA, dilution 1:2500).

Techniques: Immunofluorescence, Staining, Transfection, Western Blot, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Anti-Inflammatory Effects of miR-369-3p via PDE4B in Intestinal Inflammatory Response

doi: 10.3390/ijms25158463

Figure Lengend Snippet: Transient transfection with miR-369-3p affects the downstream PDE4B signaling pathway. ( A ) Representative blots of PKA C-α, pCREB, and CREB protein expressions after miR-369-3p mimic transfection in Raw264.7 cells without and with LPS stimulation. Western blot quantitative analysis of PKA C-α ( B ) and pCREB/CREB ( C ) expressions after miR-369-3p mimic transfection at 30 and 50 nM in unstimulated and LPS-stimulated cells. GAPDH was used as a housekeeping protein to normalize the data. Data are representative of four independent experiments. The histograms correspond to the mean ± SEM (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The primary antibodies used were rabbit mAb anti-PDE4B (ab170939, Abcam, Cambridge, UK, dilution 1:1000), rabbit mAb anti-pCREB (#9198, Cell Signaling, Technology, Danvers, MA, USA, dilution 1:1000), rabbit mAb anti-CREB (#9197, Cell Signaling, Technology, Danvers, MA, USA, dilution 1:1000), rabbit polyAb anti-PKA C-α (#4782, Cell Signaling, Technology, Danvers, MA, USA, dilution 1:1000), mouse mAb anti-GAPDH (sc-365062, Santa Cruz Biotechnology, Inc., Heidelberg, Germany, dilution 1:1000), and secondary antibodies goat anti-mouse IgG-(H+L)-HRP conjugate (170-6516, Biorad Laboratories, Hercules, CA, USA, dilution 1:500) and goat anti-rabbit IgG-(H+L)-HRP conjugate (31466, Invitrogen, Carlsbad, CA, USA, dilution 1:2500).

Techniques: Transfection, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Anti-Inflammatory Effects of miR-369-3p via PDE4B in Intestinal Inflammatory Response

doi: 10.3390/ijms25158463

Figure Lengend Snippet: PDE4B expression in UC patients. ( A ) Analysis of gene expression profiles obtained from mucosal biopsies in actively inflamed mucosa from UC patients and normal mucosa from healthy controls downloaded from the GEO database (GSE16879). ( B ) Immunohistochemical analysis of PDE4B protein expression in formalin-fixed, paraffin-embedded tissues obtained from healthy controls and patients with active UC. Original magnification, 4×. Scale bar, 100 μm (* p < 0.05).

Article Snippet: The primary antibodies used were rabbit mAb anti-PDE4B (ab170939, Abcam, Cambridge, UK, dilution 1:1000), rabbit mAb anti-pCREB (#9198, Cell Signaling, Technology, Danvers, MA, USA, dilution 1:1000), rabbit mAb anti-CREB (#9197, Cell Signaling, Technology, Danvers, MA, USA, dilution 1:1000), rabbit polyAb anti-PKA C-α (#4782, Cell Signaling, Technology, Danvers, MA, USA, dilution 1:1000), mouse mAb anti-GAPDH (sc-365062, Santa Cruz Biotechnology, Inc., Heidelberg, Germany, dilution 1:1000), and secondary antibodies goat anti-mouse IgG-(H+L)-HRP conjugate (170-6516, Biorad Laboratories, Hercules, CA, USA, dilution 1:500) and goat anti-rabbit IgG-(H+L)-HRP conjugate (31466, Invitrogen, Carlsbad, CA, USA, dilution 1:2500).

Techniques: Expressing, Immunohistochemical staining, Formalin-fixed Paraffin-Embedded

Journal: Neurotherapeutics

Article Title: A phosphodiesterase 4 (PDE4) inhibitor, amlexanox, reduces neuroinflammation and neuronal death after pilocarpine-induced seizure

doi: 10.1016/j.neurot.2024.e00357

Figure Lengend Snippet: Amlexanox's Effect on PDE4B Expression, Pro-Inflammatory Factors, and Zinc Levels in Lysosomes Following Seizures. A. PDE4B Staining: Illustrates phosphodiesterase-4B (PDE4B) expression (in red) 12 ​h after a seizure and in the sham groups. Neurons were visualized using DAPI, which was merged with PDE4B staining, highlighting the neuronal population. B. PDE4B Quantification Bar Graph: Compares the PDE4B levels between the vehicle and amlexanox groups following seizures. The analysis involved the sham vehicle (n ​= ​5), sham amlexanox (n ​= ​5), seizure vehicle (n ​= ​5), and seizure amlexanox (n ​= ​5) groups. The graph presents the mean ​± ​S.E.M. data, showing a significant difference (p ​< ​0.05) between the vehicle and amlexanox groups, as determined by the Bonferroni post hoc test following the Kruskal–Wallis test (chi-square ​= ​13.834, df ​= ​3, p ​= ​0.003). C. Zinc Staining with FluoZin-3: Demonstrates staining with FluoZin-3 in the cornu ammonis 1 (CA1) area, performed 72 ​h after a seizure. LysoTracker was used in conjunction with FluoZin-3 staining. D. Zinc Quantification Bar Graph: Displays the comparison of the zinc levels in the vehicle and amlexanox groups following seizures, involving the seizure vehicle (n ​= ​4) and seizure amlexanox (n ​= ​4) groups. The graph shows the mean ​± ​S.E.M. data, with a significant difference (p ​< ​0.05) between the groups as per the Mann–Whitney U test (z ​= ​2.309, p ​= ​0.029). E-G. Inflammatory Factor Staining: Displays the interleukin-6 (IL-6, in green) and tumor necrosis factor-α (TNF-α, in red) staining conducted 12 ​h following seizures and in sham groups. DAPI was merged with IL-6 and TNF-α staining. Inflammatory Factor Quantification Bar Graph: Compares the levels of IL-6 and TNF-α between the vehicle and amlexanox groups following seizures. The analysis included the sham vehicle (n ​= ​3), sham amlexanox (n ​= ​3), seizure vehicle (n ​= ​5), and seizure amlexanox (n ​= ​5) groups. The data are the mean ​± ​S.E.M., highlighting a significant difference (p ​< ​0.05) between the groups as established by the Bonferroni post hoc test after the Kruskal–Wallis test (IL-6: chi-square ​= ​13.412, df ​= ​3, p ​= ​0.004; TNF-α: chi-square ​= ​13.412, df ​= ​3, p ​= ​0.004). Scale bar corresponds to 20 ​μm in all images in Fig. 2 .

Article Snippet: Inc., San Antonio, TX, USA), rabbit anti- Microtubule Associated Protein 2 (MAP2, diluted 1:200; Abcam, Cambridge, UK), goat anti-Iba1 (diluted 1:500; Abcam, Cambridge, UK), mouse anti- Cluster of Differentiation 68(CD68, diluted 1:100; Bio-Rad, Hercules, CA, USA), rabbit anti- Glial fibrillary acidic protein (GFAP, diluted 1:1000; Abcam, Cambridge, UK), goat anti-Complement 3(C3, diluted 1:300; Invitrogen, Grand Island, NY, USA), rabbit anti-phosphodiesterase4B(PDE4B, diluted 1:250, FabGennix, Frisco, USA), rabbit anti-tumor necrosis factor- α (TNF-α, diluted 1:250, Abcam, Cambridge, UK), and mouse anti-interleukin-6(IL-6, diluted 1:500, Abcam, Cambridge, UK).

Techniques: Expressing, Staining, Comparison, MANN-WHITNEY

Journal: Neurotherapeutics

Article Title: A phosphodiesterase 4 (PDE4) inhibitor, amlexanox, reduces neuroinflammation and neuronal death after pilocarpine-induced seizure

doi: 10.1016/j.neurot.2024.e00357

Figure Lengend Snippet: Hypothetical Framework of the Current Study. A. Pilocarpine-Induced Seizure Schematic: This diagram provides a detailed representation of the process and critical stages in pilocarpine-induced seizures. It outlines the key steps and elements involved in the seizure induction mechanism. B. Amlexanox Administration Mechanism: This illustrates the role of amlexanox, a PDE4B inhibitor, highlighting its potential anti-inflammatory and neuroprotective effects following seizure occurrence. The diagram explains the conceptual mechanism of action of amlexanox in the context of seizure treatment and brain protection.

Article Snippet: Inc., San Antonio, TX, USA), rabbit anti- Microtubule Associated Protein 2 (MAP2, diluted 1:200; Abcam, Cambridge, UK), goat anti-Iba1 (diluted 1:500; Abcam, Cambridge, UK), mouse anti- Cluster of Differentiation 68(CD68, diluted 1:100; Bio-Rad, Hercules, CA, USA), rabbit anti- Glial fibrillary acidic protein (GFAP, diluted 1:1000; Abcam, Cambridge, UK), goat anti-Complement 3(C3, diluted 1:300; Invitrogen, Grand Island, NY, USA), rabbit anti-phosphodiesterase4B(PDE4B, diluted 1:250, FabGennix, Frisco, USA), rabbit anti-tumor necrosis factor- α (TNF-α, diluted 1:250, Abcam, Cambridge, UK), and mouse anti-interleukin-6(IL-6, diluted 1:500, Abcam, Cambridge, UK).

Techniques:

Journal: Circulation. Arrhythmia and electrophysiology

Article Title: Basal Spontaneous Firing of Rabbit Sinoatrial Node Cells is Regulated by Dual Phosphodiesterase 3 and 4 Activation

doi: 10.1161/CIRCEP.117.005896

Figure Lengend Snippet: From the left: first column, green fluorescence staining for PDE3A; second column, red fluorescence staining for marker proteins: α-actinin (A), PDE4B (B), PDE4D (C), SERCA2 (D) and PLB (E) respectively; third column merged images of PDE3A and marker proteins in panels (A)-(E). Insets in overlay show magnification of the rectangular areas in panels (A)-(E). Fourth column: intensity plots calculated along white lines in dashed rectangles. Superimposed images (Overlay) and intensity plots showed overlapping distribution of PDE3A with α-actinin, PDE4D, SERCA and PLB along Z-lines and with PDE4B beneath sarcolemma. (F) negative control had negligible fluorescence.

Article Snippet: Intact rabbit SANC were incubated with primary anti-PDE3A antibody together with either anti-α-actinin (Sigma-Aldrich, USA) or anti-PDE4B or anti-PDE4D or anti-SERCA2 (ThermoFisher, USA) or anti-PLB antibodies (Badrilla, UK).

Techniques: Fluorescence, Staining, Marker, Negative Control