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: EMBO Reports

Article Title: Bassoon controls synaptic vesicle release via regulation of presynaptic phosphorylation and cAMP

doi: 10.15252/embr.202153659

Figure Lengend Snippet: A, B Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for untreated WT and Bsn GT neurons (black and red) and cultures treated with 6‐Bnz‐cAMPS (50 μM, 1 h; grey and pink) and rolipram (1 μM, 30 min, light grey and orange). C, D Quantifications of RRP and TRP fractions in the experiment in (A) and (B). E Effect of treatment is significantly higher in Bsn GT than in WT. F, G Quantification of cAMP levels and PDE4‐associated phosphodiesterase activity assessed in hippocampal tissue of WT and Bsn GT animals. H Quantification of pSer145PDE4B to the total PDE4B ratio from the experiment in (I). I Representative immunoblot with antibody against CDK5‐dependent pSer145PDE4B and total PDE4B on hippocampal tissue from WT and Bsn GT animals. J The scheme illustrates the CDK5‐dependent regulation of cAMP hydrolysis by PDE4 and the downstream effect of PKA affecting the TRP size. The changes in this signalling detected in Bsn GT are shown; blue indicates decreased and red an increased activity or abundance. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets and corresponds to the number of imaging experiments performed on three independently prepared cultures in (C–E) or individual animals used for sample preparation in (F–H). Statistical significance was assessed using two‐way ANOVA with the Tukey's multiple comparison test in (C), (D) and (E) and the Student's t ‐test in (F), (G) and (H) and is given as: * P ≤ 0.05, ** P < 0.01, *** P < 0.001. Scale bar is 2 μm.

Article Snippet: Upon removal of free phosphate, samples containing the same protein amount were subjected to immunoprecipitation with rabbit polyclonal antibody against PDE4B (H‐56) (#sc‐25812, Santa Cruz Biotechnology) coupled to GammaBind Plus Sepharose beads (# 17‐0886‐02, GE Healthcare) for 4 h at 4°C.

Techniques: Fluorescence, Activity Assay, Western Blot, Imaging, Sample Prep, Comparison

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: