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: Tissue sections were incubated with anti-PDE4B primary antibody (MA-25677, Thermo Fisher Scientific, Waltham, MA, USA, dilution 1:1000).

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: Tissue sections were incubated with anti-PDE4B primary antibody (MA-25677, Thermo Fisher Scientific, Waltham, MA, USA, dilution 1:1000).

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: Tissue sections were incubated with anti-PDE4B primary antibody (MA-25677, Thermo Fisher Scientific, Waltham, MA, USA, dilution 1:1000).

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: Tissue sections were incubated with anti-PDE4B primary antibody (MA-25677, Thermo Fisher Scientific, Waltham, MA, USA, dilution 1:1000).

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: Tissue sections were incubated with anti-PDE4B primary antibody (MA-25677, Thermo Fisher Scientific, Waltham, MA, USA, dilution 1:1000).

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

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Phosphodiesterase 4B activation exacerbates pulmonary hypertension induced by intermittent hypoxia by regulating mitochondrial injury and cAMP/PKA/p-CREB/PGC-1α signaling.

doi: 10.1016/j.biopha.2022.114095

Figure Lengend Snippet: Fig. 1. Upregulation of PDE4B expression in Intermittent Hypoxia-induced animal models. (A) A heat map showing the top 40 differentially expressed genes in the GSE145434 dataset; each column denotes a sample, and each row represents the expression level of a gene. (B) MA plot of the GSE145434 dataset showing the fold change (y-axis) and basal expression values (x-axis) of differentially expressed genes. (C) A Venn plot of the differentially expressed genes in the GSE1909, GSE8705, and GSE145434 datasets. (D) Visualization of PDE4B expression in the three datasets. (E) Images of immunofluorescence and relative fluorescence intensity of distal pulmonary arteries of normal and CIH group rats. Double immunostaining of PDE4B (Alexa Fluor-647, red) and αSMA (Alexa Fluor-488, green) visualized smooth muscle layer. Scale bar, 10 µm. Data are shown as mean ± SE; * *P < 0.01.

Article Snippet: In addition, an anti-PDE4B antibody (1:100, CST, #72096) was used for immunofluorescence staining to observe the expression of PDE4B in lung tissue.

Techniques: Expressing, Immunofluorescence, Fluorescence, Double Immunostaining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Phosphodiesterase 4B activation exacerbates pulmonary hypertension induced by intermittent hypoxia by regulating mitochondrial injury and cAMP/PKA/p-CREB/PGC-1α signaling.

doi: 10.1016/j.biopha.2022.114095

Figure Lengend Snippet: Fig. 2. PDE4B inhibition ameliorated Intermittent Hypoxia-induced PH in rat models (A) Schematic experimental design to estimate the efficacy of AAV1.PDE4B gene inhibition on the IH-induced PH rat model. (B) Images of frozen sections of lung tissue captured by fluorescence microscopy after six weeks of AAV1.PDE4B injection; green fluorescence indicates the expression and location of AAV1 in rat lung tissue. Scale bar = 25 µm. (C) Western blot assessing the PDE4B protein expression in the control and AAV1.PDE4B groups. (D and F) Waveform diagram and quantitative analysis of RVSP (mmHg) in indicated group rats. (E) Repre sentative images of pulmonary arteries stained with H&E, Masson trichrome, and immunostaining for α-SMA and WGA in indicated group rat lung tissue. Scale bar = 25 µm. (G) The relative medial wall thickness expressed as a ratio of medial area to cross sectional area (Media/CSA). (H) Body weight of indicated rats. (I) Fulton index RV/(LV+S) is assessed in the four groups. (J) Quantitative analysis of WGA to assess hypertrophy of cardiac myocytes. (K-L) The mRNA expression of fibrosis- associated proteins (Col-1 and Fibronectin) in lungs of rats in the four groups. N = 6 rats per group. Data are shown as mean ± SE; *P < 0.05, * *P < 0.01.

Article Snippet: In addition, an anti-PDE4B antibody (1:100, CST, #72096) was used for immunofluorescence staining to observe the expression of PDE4B in lung tissue.

Techniques: Inhibition, Fluorescence, Microscopy, Injection, Expressing, Western Blot, Control, Staining, Immunostaining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Phosphodiesterase 4B activation exacerbates pulmonary hypertension induced by intermittent hypoxia by regulating mitochondrial injury and cAMP/PKA/p-CREB/PGC-1α signaling.

doi: 10.1016/j.biopha.2022.114095

Figure Lengend Snippet: Fig. 3. PDE4B deficiency ameliorated cell proliferation and mitochondrial injury in Intermittent Hypoxia-PASMCs. (A) The cell viability of PASMCs in four group is measured using a CCK8 kit. (B) The proliferation (up, Edu kit, green showed positive cells) and migration (down) are measured in PASMCs that were treated or not treated with si-PDE4B under normoxic or IH conditions, scale bars = 25 µm. (C) Quantitative measurement of Edu and migration. (D) Confocal fluorescence images of the mitochondrial membrane potential of the four indicated group cells, assessed using the JC-1 kit. The ratio of red/green fluorescence (Aggregates/Monomer) represents the degree of mitochondrial membrane potential depolarization. scale bars = 5 µm. (E) Fluorescence images of mtROS in PASMCs assessed using the mitochondrial-targeted ROS indicator—MitoSOX. MitoTracker green is used to locate mitochondria. scale bars = 5 µm. (F) Semi-quantitative analysis of relative fluorescence intensity (RFI) of JC-1 and mtROS. (G) The western blot images reflecting the protein expression of MFN2 and TOM20. N = 3 for per group. Data are shown as mean ± SE; *P < 0.05, * *P < 0.01.

Article Snippet: In addition, an anti-PDE4B antibody (1:100, CST, #72096) was used for immunofluorescence staining to observe the expression of PDE4B in lung tissue.

Techniques: Migration, Fluorescence, Membrane, Western Blot, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Phosphodiesterase 4B activation exacerbates pulmonary hypertension induced by intermittent hypoxia by regulating mitochondrial injury and cAMP/PKA/p-CREB/PGC-1α signaling.

doi: 10.1016/j.biopha.2022.114095

Figure Lengend Snippet: Fig. 4. Deficiency of PDE4B exerted its effects through the cAMP /PKA/ p-CREB/PGC-1α pathway. (A) GOBP enrichment analysis of the biological processes in differential expression genes in the GSE145434 dataset. (B-C) GSEA of second messenger and cAMP signaling pathways in the GSE145434 dataset. (D-E) The detection of cAMP concentration (pmol/ml) and PKA activity (mU/ml) in rat serum of each group using an ELISA kit. (F-G) The western blot images and quantitative analysis of protein expression of p-CREB, CREB, PGC-1α, and PPARA. N = 6 for per group rats. Data are shown as mean ± SE; *P < 0.05, * *P < 0.01.

Article Snippet: In addition, an anti-PDE4B antibody (1:100, CST, #72096) was used for immunofluorescence staining to observe the expression of PDE4B in lung tissue.

Techniques: Quantitative Proteomics, Protein-Protein interactions, Concentration Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Phosphodiesterase 4B activation exacerbates pulmonary hypertension induced by intermittent hypoxia by regulating mitochondrial injury and cAMP/PKA/p-CREB/PGC-1α signaling.

doi: 10.1016/j.biopha.2022.114095

Figure Lengend Snippet: Fig. 5. ATF4 positively regulated PDE4B expression in rat PASMCs. (A-B) The potential binding site and luciferase assay indicating the activity of PDE4B promoter with wild-type (WT) or mutant (Mut) ATF4-binding site in PASMCs transfected with pcDNA-ATF4 or si-ATF4. (C) ChIP and qPCR revealing the ATF4 enrichment and transcript levels of PDE4B in PASMCs. (D-E) Western blot and quantitative analysis reflecting that ATF4 positively regulates PDE4B expression in rat PASMCs. (F-G) The concentration of cAMP and PKA activity in PASMCs culture medium supernatant detected using an ELISA kit. (H-L) Western blot assays and quantitative analysis indicating the protein expression of target genes in PASMCs transfected with si-ATF4 or si-PDE4B and in those co-transfected. The western blots results were normalized to GAPDH. N = 3 per group in PASMCs. Data are shown as mean ± SE; *P < 0.05, * *P < 0.01, * **P < 0.001.

Article Snippet: In addition, an anti-PDE4B antibody (1:100, CST, #72096) was used for immunofluorescence staining to observe the expression of PDE4B in lung tissue.

Techniques: Expressing, Binding Assay, Luciferase, Activity Assay, Mutagenesis, Transfection, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay