Journal: Nutrients

Article Title: Integrated Metabolomics and Network Pharmacology Investigation of Cardioprotective Effects of Myricetin after 1-Week High-Intensity Exercise

doi: 10.3390/nu15061336

Figure Lengend Snippet: Effect of myricetin on the myocardial ischemic-hypoxic area and CX43 protein content. ( A ) Representative hematoxylin basic magenta picrate (HBFP) staining results. The red-stained area is the ischemic-hypoxic myocardium, and the yellow area is the normal myocardium; ( B ) representative immunofluorescence staining results. CX43 shows red fluorescence, and the nucleus shows blue fluorescence; ( C ) myocardial ischemic-hypoxic area; and ( D ) CX43 mean fluorescence density. Black arrows indicate ischemic-hypoxic myocardium. The data are presented as mean ± SD. * p < 0.05, *** p < 0.001, and ns represents no statistical difference; scale bar = 100 μm.

Article Snippet: Antigen blocking was performed after antigen retrieval with Immunol Staining Blocking Buffer (P0260, Beyotime, Shanghai, China), and sections were then incubated overnight with CX43 antibody (1:100 dilution, CST, Boston, MA, USA) at 4 °C.

Techniques: Staining, Immunofluorescence, Fluorescence

Journal: Advanced Science

Article Title: NAD+‐Boosters Improve Mitochondria Quality Control In Parkinson's Disease Models Via Mitochondrial UPR

doi: 10.1002/advs.202408503

Figure Lengend Snippet: NAD + booster NMN supplementation inhibits motor deficit and forestalls neuropathology phenotypes in MPTP‐induced mice: a) Flowchart of NMN intraperitoneal injection for treating MPTP‐induced mice model of PD. b) Plasma levels of NAD + in different groups of mice after NMN treatment (n = 4 independent samples; One‐way ANOVA). c) Striatum levels of NAD + in different groups of mice after NMN treatment (n = 4 independent samples; One‐way ANOVA). d–g) Bar plots of performance in the behavioral tests, including the open field test, pole test, rotarod test, and Suspension tests. (n = 4 independent samples; One‐way ANOVA). h,i) Levels of TH in the striatum of mice under NMN treatment by Western blot and IFC. For (h), scale bars, 2 mm (n = 4 independent samples; One‐way ANOVA). GAPDH was used as loading control for TH. j,k) Dopamine transporter levels in mice striatum and substantia nigra (n = 4). Data are shown as mean ± SEM. The P values are indicated on the graphs.

Article Snippet: MPTP and NMN were purchased from Macklin; rotenone was purchased from Aladdin; NAT was purchased from MCE; cell counting kit‐8 (lipo2000 (GK20005) and MitoSOX (GC68230) were purchased from GLPBIO; siCD38 was purchased from GenePharma; BCA protein assay kit (P0010), ATP assay kit (S0026B), NAD + /NADH assay kit with WST‐8 (S0175), mitochondrial membrane potential assay kit with JC‐1 (C2006), DAPI staining solution (C1006), antifade mounting medium (P0128M), primary antibody diluent (P0023A), RIPA lysis buffer (P0013B), PMSF (ST506), QuickBlock blocking buffer for immunol staining (P0260), QuickBlock primary antibody dilution buffer for immunol staining (P0262), and QuickBlock secondary antibody dilution buffer for immunofluorescence (P0265) were purchased from Beyotime; Annexin V‐FITC/PI kit (FXP018Pro‐050) was purchased from 4A BIOTECH; RNA simple total RNA kit (DP419) was purchased from TIANGEN; qRT Master Mix (RTQ‐100) and qPCR Master Mix (QST‐100) were purchased from Toroivd; protein‐free rapid blocking buffer (PS108) was purchased from Epizyme; and paraformaldehyde (BL539A) and TRIS_EDTA antigen retrieval solution (50X) (BL617A) were purchased from Biosharp.

Techniques: Injection, Clinical Proteomics, Suspension, Western Blot, Control

Journal: Advanced Science

Article Title: NAD+‐Boosters Improve Mitochondria Quality Control In Parkinson's Disease Models Via Mitochondrial UPR

doi: 10.1002/advs.202408503

Figure Lengend Snippet: Omics signatures of the striatum with NMN in MPTP‐induced PD mice: a,b) differential genes for the MPTP and Control groups are shown in the volcano plots based on ‐log10 ( P value) and log 2 FC, whereas MPTP+NMN and MPTP groups are displayed in b in the same way. c) Heatmap of differential genes in Control, MPTP, and MPTP+NMN groups. d) PCA analysis of Control, MPTP, and MPTP+NMN groups (n = 3). e) KEGG analysis of differential genes for the MPTP versus control group, and the MPTP + NMN versus MPTP group, and co‐enrichment of the pathways based on ‐log 10 ( P value) and log 2 FC of related genes within the crucial KEGG pathways are shown in bar graphs. f) GO enrichment analysis of differential genes in the MPTP versus Control group, and MPTP + NMN versus MPTP group, and the core pathways are shown based on up‐ and down‐regulated normalized values and ‐log 10 ( P value). g–i) Mfuzz trend analysis of Control, MPTP, and MPTP + NMN groups, and their expression trends were visualized in g, and all genes in different groups were plotted as scatter plots in i based on their MPTP + NMN/MPTP expression ratios and expression ratios with the control group for trends 2 and 5. h) The GSEA enrichment results of MPTP and MPTP + NMN groups according to mitochondria‐related pathways, and energy metabolism‐related pathways were visualized in radar plots. P values and R coefficients are derived from Spearman correlations. Data are shown as mean ± SEM. The P values are indicated on the graphs.

Article Snippet: MPTP and NMN were purchased from Macklin; rotenone was purchased from Aladdin; NAT was purchased from MCE; cell counting kit‐8 (lipo2000 (GK20005) and MitoSOX (GC68230) were purchased from GLPBIO; siCD38 was purchased from GenePharma; BCA protein assay kit (P0010), ATP assay kit (S0026B), NAD + /NADH assay kit with WST‐8 (S0175), mitochondrial membrane potential assay kit with JC‐1 (C2006), DAPI staining solution (C1006), antifade mounting medium (P0128M), primary antibody diluent (P0023A), RIPA lysis buffer (P0013B), PMSF (ST506), QuickBlock blocking buffer for immunol staining (P0260), QuickBlock primary antibody dilution buffer for immunol staining (P0262), and QuickBlock secondary antibody dilution buffer for immunofluorescence (P0265) were purchased from Beyotime; Annexin V‐FITC/PI kit (FXP018Pro‐050) was purchased from 4A BIOTECH; RNA simple total RNA kit (DP419) was purchased from TIANGEN; qRT Master Mix (RTQ‐100) and qPCR Master Mix (QST‐100) were purchased from Toroivd; protein‐free rapid blocking buffer (PS108) was purchased from Epizyme; and paraformaldehyde (BL539A) and TRIS_EDTA antigen retrieval solution (50X) (BL617A) were purchased from Biosharp.

Techniques: Control, Expressing, Derivative Assay

Journal: Advanced Science

Article Title: NAD+‐Boosters Improve Mitochondria Quality Control In Parkinson's Disease Models Via Mitochondrial UPR

doi: 10.1002/advs.202408503

Figure Lengend Snippet: MPTP‐induced reactive gliosis and synaptic disruption were attenuated by NMN treatment: a) RT‐qPCR analysis revealed that the expression of genes such as Il‐1β , II‐6 , Tnf , Nos2 , Ccl2 , and Ccl2 (n = 3 independent samples; One‐way ANOVA). b–f) Effects of NMN on the expression level of IBA1 and GFAP (n = 3 independent samples; One‐way ANOVA). β‐actin was used as loading control. g,h,j) Effects of NMN on the expression level of synaptophysin and PSD95 (n = 5 independent samples; One‐way ANOVA). β‐actin was used as loading control. i,k) Effects of NMN on the expression level of NeuN (n = 6 independent samples; One‐way ANOVA). Data are shown as mean ± s.e.m. The P values are indicated on the graphs. NS, not significant.

Article Snippet: MPTP and NMN were purchased from Macklin; rotenone was purchased from Aladdin; NAT was purchased from MCE; cell counting kit‐8 (lipo2000 (GK20005) and MitoSOX (GC68230) were purchased from GLPBIO; siCD38 was purchased from GenePharma; BCA protein assay kit (P0010), ATP assay kit (S0026B), NAD + /NADH assay kit with WST‐8 (S0175), mitochondrial membrane potential assay kit with JC‐1 (C2006), DAPI staining solution (C1006), antifade mounting medium (P0128M), primary antibody diluent (P0023A), RIPA lysis buffer (P0013B), PMSF (ST506), QuickBlock blocking buffer for immunol staining (P0260), QuickBlock primary antibody dilution buffer for immunol staining (P0262), and QuickBlock secondary antibody dilution buffer for immunofluorescence (P0265) were purchased from Beyotime; Annexin V‐FITC/PI kit (FXP018Pro‐050) was purchased from 4A BIOTECH; RNA simple total RNA kit (DP419) was purchased from TIANGEN; qRT Master Mix (RTQ‐100) and qPCR Master Mix (QST‐100) were purchased from Toroivd; protein‐free rapid blocking buffer (PS108) was purchased from Epizyme; and paraformaldehyde (BL539A) and TRIS_EDTA antigen retrieval solution (50X) (BL617A) were purchased from Biosharp.

Techniques: Disruption, Quantitative RT-PCR, Expressing, Control

Journal: Advanced Science

Article Title: NAD+‐Boosters Improve Mitochondria Quality Control In Parkinson's Disease Models Via Mitochondrial UPR

doi: 10.1002/advs.202408503

Figure Lengend Snippet: Treatment with NMN remodels metabolic pathways of the striatum in MPTP‐induced PD mice: a) OPLS‐DA analysis of Control, MPTP, and MPTP + NMN groups (n = 3). b) Metabolites for MPTP + NMN and MPTP groups based on ‐log 10 ( P value) and log 2 FC are presented in the volcano plots. c,d) Mfuzz trend analysis of metabolites obtained from positive and negative ion patterns for different groups is presented in (c), and integration and visualization of critical metabolite subclusters' expression levels are plotted in the heatmap (d). e) MSEA enrichment analysis of differential metabolites in the MPTP + NMN vs MPTP group, and the pathway with the highest enrichment level was selected and visualized based on its p‐value. f) GO pathway enrichment analysis of differential metabolites in the MPTP + NMN vs MPTP group, and metabolites involved in multiple pathways were plotted as chordal plots based on their pathway interactions. g) WCGNA of control, MPTP, and MPTP + NMN groups, where the cluster dendrogram represents the metabolite groups identified by WGCNA. Meanwhile, the heatmap represents the correlation of different metabolite modules with NMN and MPTP. The critical metabolites in the Yellow, Grey, and Green modules are further analyzed in supple Figure . h) Combined metabolomics and transcriptomics analysis of Control, MPTP, and MPTP + NMN groups, where dots represent metabolite abundance, lines represent transcriptional expression of the enzymes, and the color differences indicate the degree of their high expression level in the MPTP or NMN groups. Then, peak plots indicate the respective degree of expression of all detected metabolites.

Article Snippet: MPTP and NMN were purchased from Macklin; rotenone was purchased from Aladdin; NAT was purchased from MCE; cell counting kit‐8 (lipo2000 (GK20005) and MitoSOX (GC68230) were purchased from GLPBIO; siCD38 was purchased from GenePharma; BCA protein assay kit (P0010), ATP assay kit (S0026B), NAD + /NADH assay kit with WST‐8 (S0175), mitochondrial membrane potential assay kit with JC‐1 (C2006), DAPI staining solution (C1006), antifade mounting medium (P0128M), primary antibody diluent (P0023A), RIPA lysis buffer (P0013B), PMSF (ST506), QuickBlock blocking buffer for immunol staining (P0260), QuickBlock primary antibody dilution buffer for immunol staining (P0262), and QuickBlock secondary antibody dilution buffer for immunofluorescence (P0265) were purchased from Beyotime; Annexin V‐FITC/PI kit (FXP018Pro‐050) was purchased from 4A BIOTECH; RNA simple total RNA kit (DP419) was purchased from TIANGEN; qRT Master Mix (RTQ‐100) and qPCR Master Mix (QST‐100) were purchased from Toroivd; protein‐free rapid blocking buffer (PS108) was purchased from Epizyme; and paraformaldehyde (BL539A) and TRIS_EDTA antigen retrieval solution (50X) (BL617A) were purchased from Biosharp.

Techniques: Control, Expressing

Journal: Journal of Nanobiotechnology

Article Title: Poria cocos-derived exosome-like nanoparticles ameliorate lymphedema by reprogramming fibroblast metabolism via enhanced TCA cycle flux

doi: 10.1186/s12951-026-04200-z

Figure Lengend Snippet: PcELNs reverse TGF-β-induced pro-fibrotic phenotypes in HFF cells by suppressing migration and invasion while enhancing collagenolytic activity. ( A ) Schematic diagram of the sequential treatment: HFF cells were first pre-treated with TGF-β (10 ng/ml) for 24 hours to induce a pro-fibrotic state, followed by induction with PcELNs. ( B) Representative images and quantification of the scratch healing assay at 0 and 12 hours, assessing cell migration (n = 5 per group, scale bar = 200 μm). ( C ) Typical confocal images and quantification of invasive foot formation (white: DAPI, red: CTTN, blue: F-actin, green: MMP9, n = 20 per group, scale bar = 20 μm). ( D ) Cell migration and invasion evaluated by Transwell assay (n = 5 per each group, scale bar = 100 μm). ( E ) Representative confocal images of EMT markers (E-CAD, N-CAD, VIM, α-SMA) and collagenases (MMP1, MMP3) in HFF cells after induction by TGF-β and PcELNs (scale bar = 25 μm). ( F ) Western blot analysis of key proteins related to EMT (E-CAD, N-CAD, α-SMA, VIM) and collagen degradation (MMP1, MMP3). (G ) Quantification of EMT markers (E-CAD, N-CAD, VIM, α-SMA) and collagen degradation (MMP1, MMP3) at the protein and transcription levels (n = 4 per each group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. All values are presented as the mean ± SD

Article Snippet: Mito-Tracker Deep Red FM (C1032-50 μg), Mito-Tracker Green (C1048), MitoSOTM Red (S0061S), Rhod-2 AM (S1062S), Hoechst 33,342 (C1022), Lyso-Tracker Green (C1047S), Lyso-Tracker Red (C1046), Actin-Tracker Red-Rhodamine (C2207S), Tubulin-Tracker Deep Red Staining Kit for Living Cells (C2215S), Antifade Mounting Medium with DAPI (P0131-25 ml), RIPA Lysis Buffer (P0013B), 2 mm Ceramic Beads for Tissue Homogenizer, DNase, RNase & Protease free (F6652), 4 mm Ceramic Beads for Tissue Homogenizer, DNase, RNase & Protease free (F6654), Microcentrifuge Tubes for Tissue Homogenizer, Thickened, 2 ml (F6646-1bag), QuickBlockTM Protein-Free Blocking Buffer for Western Blot (P0240-500 ml), QuickBlockTM Blocking Buffer for Immunol Staining (P0260), BeyoGoldTM Transwell Permeable Supports (FTW164-48Ins) were purchased from Beyotime Biotech Inc (Shanghai, China).

Techniques: Migration, Activity Assay, Transwell Assay, Western Blot

Journal: Frontiers in immunology

Article Title: Repression of JAK2-STAT1 and PD-L1 by CEP-33779 ameliorates the LPS-induced decline in phagocytic activity of alveolar macrophages and mitigates lung injury in mice.

doi: 10.3389/fimmu.2024.1472425

Figure Lengend Snippet: FIGURE 1 Immunohistochemistry revealed an increased expression of PD-L1 on AMs in the lung tissue of a mouse model with ALI. (A) The wet-to-dry weight ratio of lung tissues was measured in both the control group and the ALI model group. (B) Protein concentration in BALF was quantified. (C) Lung injury score was determined. (D) Representative photomicrographs of lung tissues stained with hematoxylin and eosin (H&E) were captured. (E, F) The expression level of PD-L1 on AMs in lung tissue was evaluated by IHC and double immunofluorescence staining. The results showed that the expression of PD-L1 on inflammatory cells in the lung tissue of ALI mice was significantly increased, and the expression of PD-L1 on AMs was the strongest. Scale bars indicate 100 mm, and 50 mm. The data are presented as Mean ± SE with n = 5 samples per group. Statistical analysis showed ****P < 0.0001.

Article Snippet: The sections were then incubated with QuickBlockTM Blocking Buffer for Immunol Staining (Beyotime, cat#: P0260) for 15 minutes and stained frontiersin.org overnight at 4°C with a primary antibody against PD-L1 (diluted 1:200, Proteintech, cat#:66248-1-Ig).

Techniques: Immunohistochemistry, Expressing, Control, Protein Concentration, Staining

Journal: Frontiers in immunology

Article Title: Repression of JAK2-STAT1 and PD-L1 by CEP-33779 ameliorates the LPS-induced decline in phagocytic activity of alveolar macrophages and mitigates lung injury in mice.

doi: 10.3389/fimmu.2024.1472425

Figure Lengend Snippet: FIGURE 2 CEP-33779 concentration-dependently suppressed the activation of the JAK2-STAT1 pathway and upregulation of PD-L1 on freshly isolated AMs in an LPS-induced ALI mouse model as shown by immunofluorescence staining. Cells in BALF were separated from each group of mice for immunofluorescence staining, and the expression of p-JAK2, p-STAT1, and PD-L1 was visualized using laser confocal microscopy. Blue fluorescence corresponds to DAPI-stained nuclei; Red fluorescence represents CD11c stained with CD11c antibodies, marking alveolar macrophages. Green fluorescence indicates staining for phosphorylated JAK2 antibodies, phosphorylated STAT1 antibody and PD-L1 antibodies respectively. All laser confocal microscopy photos were captured using a 40x oil mirror. Scale bars indicate 20 mm.

Article Snippet: The sections were then incubated with QuickBlockTM Blocking Buffer for Immunol Staining (Beyotime, cat#: P0260) for 15 minutes and stained frontiersin.org overnight at 4°C with a primary antibody against PD-L1 (diluted 1:200, Proteintech, cat#:66248-1-Ig).

Techniques: Concentration Assay, Activation Assay, Isolation, Staining, Expressing, Confocal Microscopy

Journal: Frontiers in immunology

Article Title: Repression of JAK2-STAT1 and PD-L1 by CEP-33779 ameliorates the LPS-induced decline in phagocytic activity of alveolar macrophages and mitigates lung injury in mice.

doi: 10.3389/fimmu.2024.1472425

Figure Lengend Snippet: FIGURE 3 The activation of the JAK2-STAT1 signaling pathway and PD-L1 expression in MH-S cells was induced by LPS. MH-S cells were stimulated with LPS (1 mg/ml) for different durations: 0, 15, 30, 60, and 120 minutes. Western blot analysis was performed to detect the phosphorylation levels and total protein levels of JAK2 and STAT1. A significant increase in phosphorylation of JAK2 and STAT1 was observed at 15 minutes after LPS stimulation (A, B). PD-L1 expression was enhanced after LPS stimulation for 24 and 48 hours (C). Quantitative analysis of the intensity of pJAK2, pSTAT1, and PD-L1 bands (D–F). ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: The sections were then incubated with QuickBlockTM Blocking Buffer for Immunol Staining (Beyotime, cat#: P0260) for 15 minutes and stained frontiersin.org overnight at 4°C with a primary antibody against PD-L1 (diluted 1:200, Proteintech, cat#:66248-1-Ig).

Techniques: Activation Assay, Expressing, Western Blot, Phospho-proteomics

Journal: Frontiers in immunology

Article Title: Repression of JAK2-STAT1 and PD-L1 by CEP-33779 ameliorates the LPS-induced decline in phagocytic activity of alveolar macrophages and mitigates lung injury in mice.

doi: 10.3389/fimmu.2024.1472425

Figure Lengend Snippet: FIGURE 4 The effect of CEP-33779 and Fludarabine on p-JAK2, p-STAT1, and PD-L1 expression in MH-S cells was analyzed using western blot and immunofluorescence. MH-S cells were pre-treated with varying concentrations (10, 100, 1000, and 3000 nM) of CEP-33779 for one hour before stimulation with 1 mg/ml of LPS for two hours. Subsequently, western blot analysis was performed to detect the phosphorylation levels and total protein levels of JAK2 (A) and STAT1 (B). Quantitative analysis was conducted to determine the levels of phosphorylated JAK2, phosphorylated STAT1 and PD-L1 expression (C, D, I, J, L). Representative confocal microscopy images from three independent experiments are shown in panels (E–G). MH-S cells in the LPS group were stimulated with 1 mg/ml LPS for either 2 hours (for p-JAK2 and p-STAT1 expression) or 24 hours (for PD-L1 expression). In the LPS+CEP-33779 group, MH-S cells were pretreated with 3000 nM CEP-33779 for either 1 hour or 2 hours, followed by co- treatment with 1 mg/ml LPS and 3000 nM CEP-33779 for either 2 or 24 hours. Blue fluorescence indicates cell nuclei stained with DAPI; (E, F) show green fluorescence representing p-JAK2 and p-STAT1 with nuclear translocation expression respectively; (G) shows red fluorescence indicating PD- L1. All laser confocal microscopy photos were taken using a 40x oil mirror. (H, K) cells are pretreated with 50 mM of the STAT1 inhibitor fludarabine (MCE, cat number: 21679-14-1) or without it for 24 h. Subsequently, cells are treated with 1 mg/ml LPS for 30 min to detect the levels of p-JAK2 and p-STAT1. In addition, the protein expression levels of PD-L1 are detected by pretreatment with or without 50 mM fludarabine for 24 h followed by treatment with 1 mg/ml LPS for 48 h. The scale bar is 20 mm. The results are presented as Mean ± SE from three independent experiments (ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Article Snippet: The sections were then incubated with QuickBlockTM Blocking Buffer for Immunol Staining (Beyotime, cat#: P0260) for 15 minutes and stained frontiersin.org overnight at 4°C with a primary antibody against PD-L1 (diluted 1:200, Proteintech, cat#:66248-1-Ig).

Techniques: Expressing, Western Blot, Phospho-proteomics, Confocal Microscopy, Staining, Translocation Assay