Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Gut Metabolite Indole-3-Propionic Acid Regulates Macrophage Autophagy Through PPT1 Inhibiting Aging-Related Myocardial Fibrosis.

doi: 10.1002/advs.202501070

Figure Lengend Snippet: Figure 4. PPT1 expression is upregulated in senescent macrophages. A) Volcano plot of differentially expressed proteins of Young and Aged groups in rats heart. B) KEGG enrichment top 20. C) Heat map of differentially expressed proteins in the lysosomal pathway. D,E) Representative images and statistical analysis of IHC staining with PPT1 antibody. Magnification: 400×, scale bar = 100 μm. F) Statistical analysis of PPT1 mRNA level. G) Representative images and statistical analysis of PPT1 protein level. H) UMAP plots of heart macrophage from 27 healthy donors. I) A heatmap showing the markers ≈7 types of macrophages. J) Stacked graph of cell proportions. K) Violin plots of inflammatory response scores in different subgroups. L) Violin plots of the M1 scores of different macrophage subsets. M) Violin plots of PPT1 expression in different monocyte-macrophage subsets. N) Inflammatory response score. O) M1 score. P) Representative figure of the immunofluorescence of CD68, PPT1, and DAPI in heart. Magnification: 200×, scale bar = 20 μm. Q,R) Representative immunofluorescence images and statistical analysis of PPT1 intensity. Magnification: 200×, scale bar = 20 μm. S) Statistical analysis of PPT1 mRNA level in BMDM. T) Representative graphs and statistical analysis of PPT1 at protein level in BMDM. (n = 3–6, data are expressed as mean ± SEM, *p < 0.05, **p < 0.01 vs the Young group).

Article Snippet: The antibody resources are as follows: ANP primary antibody (Proteintech, 27426-1-Ig, USA), β-tubulin primary antibody (Proteintech, 10094-1-Ig, USA), STING primary antibody (Proteintech, 19851-1-Ig, USA), AKT primary antibody (Proteintech, 60203-2-Ig, USA), PPT1 primary antibody (Proteintech, 29653-1-Ig, USA), COL1A1 primary antibody (Proteintech, 66761-1-Ig, USA), TNF-α primary antibody (Proteintech, 60291-1-Ig, USA), cGAS primary antibody (ABclonal, A8335, China), PI3K primary antibody (Affinity Biosciences, AF6241, China), p-PI3K primary antibody (CST, 4228S, USA), p-AKT primary antibody (Affinity Biosciences, AF0016, China), MCP-1 primary Adv.

Techniques: Expressing, Immunohistochemistry

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Gut Metabolite Indole-3-Propionic Acid Regulates Macrophage Autophagy Through PPT1 Inhibiting Aging-Related Myocardial Fibrosis.

doi: 10.1002/advs.202501070

Figure Lengend Snippet: Figure 5. Transgenic knockout of macrophage PPT1 improves cardiac inflammatory infiltration and myocardial fibrosis in D-gal induce-aged mice. A) Schematic of the experimental design. B) Representative echocardiographic graphs. C–J) Echocardiographic measurements of LVEF, LVFS, LVIDd, LVIDs, LVPWd, LVPWs, IVSd, and IVSs. K) H&E staining of left ventricles. Magnification: 200×, scale bar = 50 μm. L) Masson’s staining of left ventricles. Magnification: 200×, scale bar = 50 μm. M,N) Representative images of IHC staining with MCP-1 and 𝛼-SMA antibody. Magnification: 200×, scale bar = 50 μm. O) Statistical graphs of Masson’s staining. P,Q) Statistical graphs of IHC staining with MCP-1 and 𝛼-SMA antibody. R) Representative images of the immunofluorescence of CD68, PPT1, and DAPI in mice heart. Magnification: 200×, scale bar = 20 μm. S) Statistical analysis of COL3A1, COL1A1, PPT1, IL-6, and TNF-𝛼mRNA level. T,U) Representative images and statistical analysis of COL3A1, COL1A1, PPT1, IL-6, and TNF-𝛼at protein level. (n = 5–6, data are expressed as mean ± SEM, **p < 0.01 vs the WT group; #p < 0.05, ##p < 0.01 vs the D-gal group).

Article Snippet: The antibody resources are as follows: ANP primary antibody (Proteintech, 27426-1-Ig, USA), β-tubulin primary antibody (Proteintech, 10094-1-Ig, USA), STING primary antibody (Proteintech, 19851-1-Ig, USA), AKT primary antibody (Proteintech, 60203-2-Ig, USA), PPT1 primary antibody (Proteintech, 29653-1-Ig, USA), COL1A1 primary antibody (Proteintech, 66761-1-Ig, USA), TNF-α primary antibody (Proteintech, 60291-1-Ig, USA), cGAS primary antibody (ABclonal, A8335, China), PI3K primary antibody (Affinity Biosciences, AF6241, China), p-PI3K primary antibody (CST, 4228S, USA), p-AKT primary antibody (Affinity Biosciences, AF0016, China), MCP-1 primary Adv.

Techniques: Transgenic Assay, Knock-Out, Staining, Immunohistochemistry

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Gut Metabolite Indole-3-Propionic Acid Regulates Macrophage Autophagy Through PPT1 Inhibiting Aging-Related Myocardial Fibrosis.

doi: 10.1002/advs.202501070

Figure Lengend Snippet: Figure 6. IPA inhibits PPT1 expression and reduces the secretion of inflammatory factors in aging macrophages. A,B) Representative images and statistical graphs of the immunofluorescence of iNOS. Magnification: 200×, scale bar = 20 μm. C) Statistical analysis of IL-6 and TNF-𝛼at mRNA level. D,E) Representative images and statistical analysis of IL-6 and TNF-𝛼at protein level. F,G) Representative images and statistical graphs of the immunofluorescence of iNOS. Magnification: 200×, scale bar = 20 μm. H) Statistical analysis of IL-6 and TNF-𝛼at mRNA level. I,J) Representative images and statistical analysis of IL-6 and TNF-𝛼at protein level. (n = 3–6, data are expressed as mean ± SEM, **p < 0.01 vs the Young or Vector group; #p < 0.05, ##p < 0.01 vs the Aged or oe-PPT1 group).

Article Snippet: The antibody resources are as follows: ANP primary antibody (Proteintech, 27426-1-Ig, USA), β-tubulin primary antibody (Proteintech, 10094-1-Ig, USA), STING primary antibody (Proteintech, 19851-1-Ig, USA), AKT primary antibody (Proteintech, 60203-2-Ig, USA), PPT1 primary antibody (Proteintech, 29653-1-Ig, USA), COL1A1 primary antibody (Proteintech, 66761-1-Ig, USA), TNF-α primary antibody (Proteintech, 60291-1-Ig, USA), cGAS primary antibody (ABclonal, A8335, China), PI3K primary antibody (Affinity Biosciences, AF6241, China), p-PI3K primary antibody (CST, 4228S, USA), p-AKT primary antibody (Affinity Biosciences, AF0016, China), MCP-1 primary Adv.

Techniques: Expressing, Plasmid Preparation

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Gut Metabolite Indole-3-Propionic Acid Regulates Macrophage Autophagy Through PPT1 Inhibiting Aging-Related Myocardial Fibrosis.

doi: 10.1002/advs.202501070

Figure Lengend Snippet: Figure 7. IPA inhibits PPT1 in aged macrophages and alleviates collagen deposition in fibroblasts. A) Schematic of the experimental design. B,C) Representative figures and statistical graphs of the immunofluorescence of 𝛼-SMA. Magnification: 200×, scale bar = 20 μm. D) Statistical analysis of COL3A1, COL1A1 and 𝛼-SMA at mRNA level. E,F) Representative images and statistical analysis of COL3A1, COL1A1 and 𝛼-SMA at protein level. G,H) Representative Figure and statistical graphs of the immunofluorescence of 𝛼-SMA. Magnification: 200×, scale bar = 20 μm. I) Statistical analysis of COL3A1, COL1A1 and 𝛼-SMA at mRNA level. J,K) Representative figures and statistical analysis of COL3A1, COL1A1 and 𝛼-SMA at protein level. (n = 3– 6, data are expressed as mean ± SEM, *p < 0.5, **p < 0.01 vs the Young–CM or Vector–CM group; #p < 0.5, ##p < 0.01 vs the Aged–CM or oe-PPT1–CM group).

Article Snippet: The antibody resources are as follows: ANP primary antibody (Proteintech, 27426-1-Ig, USA), β-tubulin primary antibody (Proteintech, 10094-1-Ig, USA), STING primary antibody (Proteintech, 19851-1-Ig, USA), AKT primary antibody (Proteintech, 60203-2-Ig, USA), PPT1 primary antibody (Proteintech, 29653-1-Ig, USA), COL1A1 primary antibody (Proteintech, 66761-1-Ig, USA), TNF-α primary antibody (Proteintech, 60291-1-Ig, USA), cGAS primary antibody (ABclonal, A8335, China), PI3K primary antibody (Affinity Biosciences, AF6241, China), p-PI3K primary antibody (CST, 4228S, USA), p-AKT primary antibody (Affinity Biosciences, AF0016, China), MCP-1 primary Adv.

Techniques: Plasmid Preparation

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Gut Metabolite Indole-3-Propionic Acid Regulates Macrophage Autophagy Through PPT1 Inhibiting Aging-Related Myocardial Fibrosis.

doi: 10.1002/advs.202501070

Figure Lengend Snippet: Figure 8. IPA inhibits PPT1 in elderly macrophages in vitro and reduces myocardial fibrosis in vivo. A) Schematic of the experimental design. B,C) Rep- resentative graphs and statistical graph of flow cytometry. D) Representative echocardiographic graphs. E–L) Echocardiographic measurements of LVEF, LVFS, LVIDd, LVIDs, LVPWd, LVPWs, IVSd, and IVSs. M) H&E staining of left ventricles. Magnification: 200×, scale bar = 50 μm. N) Masson’s staining of left ventricles. Magnification: 200×, scale bar = 50 μm. O,P) Representative images of IHC staining with MCP-1 and 𝛼-SMA antibody. Magnification: 200×, scale bar = 50 μm. Q) Statistical graphs of Masson’s staining. R,S) Statistical graphs of IHC staining with MCP-1 and 𝛼-SMA antibody. T) Statistical analysis of COL3A1, COL1A1, IL-6, and TNF-𝛼at mRNA level. U,V) Representative images and statistical analysis of COL3A1, COL1A1, IL-6, and TNF-𝛼 at protein level. (n = 3–6, data are expressed as mean ± SEM, *p < 0.05, **p < 0.01 vs the Young group; #p < 0.05, ##p < 0.01 vs the Aged group; &p < 0.05, &&p < 0.01 vs the Aged+sh-PPT1 group).

Article Snippet: The antibody resources are as follows: ANP primary antibody (Proteintech, 27426-1-Ig, USA), β-tubulin primary antibody (Proteintech, 10094-1-Ig, USA), STING primary antibody (Proteintech, 19851-1-Ig, USA), AKT primary antibody (Proteintech, 60203-2-Ig, USA), PPT1 primary antibody (Proteintech, 29653-1-Ig, USA), COL1A1 primary antibody (Proteintech, 66761-1-Ig, USA), TNF-α primary antibody (Proteintech, 60291-1-Ig, USA), cGAS primary antibody (ABclonal, A8335, China), PI3K primary antibody (Affinity Biosciences, AF6241, China), p-PI3K primary antibody (CST, 4228S, USA), p-AKT primary antibody (Affinity Biosciences, AF0016, China), MCP-1 primary Adv.

Techniques: In Vitro, In Vivo, Cytometry, Staining, Immunohistochemistry

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Gut Metabolite Indole-3-Propionic Acid Regulates Macrophage Autophagy Through PPT1 Inhibiting Aging-Related Myocardial Fibrosis.

doi: 10.1002/advs.202501070

Figure Lengend Snippet: Figure 10. cGAS-STING pathway is involved in IPA-induced inhibition of PPT1. A) Statistical graph of cGAS and STING mRNA levels in macrophages. B,C) Representative images and statistical graph of cGAS and STING at protein levels in macrophages. D,E) Representative images and statistical graph of cGAS and STING at protein levels in vitro. F) Schematic diagram of IPA. G–J) Representative images and statistical graph of PPT1 immunofluorescence intensity in M1 macrophages after treated with RU.521 or C176. Magnification: 200×, scale bar = 20 μm. K,L) Representative images and statistical graph of PPT1 protein levels in M1 macrophages after treated with RU.521 or C176. M) Graphical abstract. (n = 3–6, data are expressed as mean ± SEM, *p < 0.05, **p < 0.01 vs the Young or Control group; #p < 0.05, ##p < 0.01 vs the Aged or LPS/IFN-𝛾group).

Article Snippet: The antibody resources are as follows: ANP primary antibody (Proteintech, 27426-1-Ig, USA), β-tubulin primary antibody (Proteintech, 10094-1-Ig, USA), STING primary antibody (Proteintech, 19851-1-Ig, USA), AKT primary antibody (Proteintech, 60203-2-Ig, USA), PPT1 primary antibody (Proteintech, 29653-1-Ig, USA), COL1A1 primary antibody (Proteintech, 66761-1-Ig, USA), TNF-α primary antibody (Proteintech, 60291-1-Ig, USA), cGAS primary antibody (ABclonal, A8335, China), PI3K primary antibody (Affinity Biosciences, AF6241, China), p-PI3K primary antibody (CST, 4228S, USA), p-AKT primary antibody (Affinity Biosciences, AF0016, China), MCP-1 primary Adv.

Techniques: Inhibition, In Vitro, Control

Journal: Data in Brief

Article Title: Quantitative analysis of PPT1 interactome in human neuroblastoma cells

doi: 10.1016/j.dib.2015.05.016

Figure Lengend Snippet: Global network analysis of PPT1 IP. Functional coupling of PPT1 IP was analysed by FunCoup database v.3.0 ( http://FunCoup.sbc.su.se/ ). Based on 24 input genes (including PPT1 and PPT1 IP), 65 functionally coupled pairs with a very high confidence score (>0.8, scale 0−1) were identified. Dots, dashed and solid lines; indicate scores between (0.82–0.89), (0.9–0.95) and (>0.95), respectively. Details are provided in , Supporting Information.

Article Snippet: Membranes were blocked with 3% BSA in TBST buffer (0.01 M Tris–HCl, pH 8.0, 0.15 M NaCl, and 0.1% Tween 20) and incubated with the following primary Abs: rabbit polyclonal anti-human-PPT1 (1:500) , rabbit polyclonal anti-PPT1 (HPA021546, 1:1000) (Sigma-Aldrich Finland Oy); mouse monoclonal anti-VCP [#612182] (1:1000) (BD Transduction Laboratories TM ); mouse monoclonal anti-ATP5B [clone 3D5AB1] (A21351, 1:1000) (Molecular Probes/Life Technologies Europe BV, Espoo, Finland); rabbit polyclonal anti-DBH (NBP1-31386, 1:1500) (Novus Biologicals, Cambridge, UK); mouse monoclonal anti-Myc [9E10] (ab32, 1:1500) and mouse monoclonal anti-β-actin [AC15] (A1978, 1:2000) (Sigma-Aldrich Finland Oy).

Techniques: Functional Assay

Journal: Data in Brief

Article Title: Quantitative analysis of PPT1 interactome in human neuroblastoma cells

doi: 10.1016/j.dib.2015.05.016

Figure Lengend Snippet: PPT1 IP association terms network. PPT1 interacting partners served as inputs into Ingenuity Pathways analyses ( https://analysis.ingenuity.com/ ). Three terms: inhibition of organismal death ( Z -score −3.109), Movement disorders ( Z -score −2.209) and concentration of lipid ( Z -score −2.133) were predicted to be altered in the PPT1 network.

Article Snippet: Membranes were blocked with 3% BSA in TBST buffer (0.01 M Tris–HCl, pH 8.0, 0.15 M NaCl, and 0.1% Tween 20) and incubated with the following primary Abs: rabbit polyclonal anti-human-PPT1 (1:500) , rabbit polyclonal anti-PPT1 (HPA021546, 1:1000) (Sigma-Aldrich Finland Oy); mouse monoclonal anti-VCP [#612182] (1:1000) (BD Transduction Laboratories TM ); mouse monoclonal anti-ATP5B [clone 3D5AB1] (A21351, 1:1000) (Molecular Probes/Life Technologies Europe BV, Espoo, Finland); rabbit polyclonal anti-DBH (NBP1-31386, 1:1500) (Novus Biologicals, Cambridge, UK); mouse monoclonal anti-Myc [9E10] (ab32, 1:1500) and mouse monoclonal anti-β-actin [AC15] (A1978, 1:2000) (Sigma-Aldrich Finland Oy).

Techniques: Inhibition, Concentration Assay

Journal: Data in Brief

Article Title: Quantitative analysis of PPT1 interactome in human neuroblastoma cells

doi: 10.1016/j.dib.2015.05.016

Figure Lengend Snippet: PPT1 interaction network. PPT1 interaction partners were linked to PPT1 using known physical and genetic interactions, pathway data and utilising knowledge of predicted interactions. The network was filtered according to GO BP criteria using GeneMANIA algorithm ( www.genemania.org ). Overall, 74 nodes of the network could be connected using 88 physical and 69 genetic interactions, 124 pathway and 133 predicted links. Pathway information from Pathway commons (Consolidated-Pathways-2013) was used to facilitate additional 16 links with varying weights (0.15–16.83%) to the PPT1 network. Nodes enriched with selected GO BP terms are indicated. Detailed information about the parameters of the network is presented in .

Article Snippet: Membranes were blocked with 3% BSA in TBST buffer (0.01 M Tris–HCl, pH 8.0, 0.15 M NaCl, and 0.1% Tween 20) and incubated with the following primary Abs: rabbit polyclonal anti-human-PPT1 (1:500) , rabbit polyclonal anti-PPT1 (HPA021546, 1:1000) (Sigma-Aldrich Finland Oy); mouse monoclonal anti-VCP [#612182] (1:1000) (BD Transduction Laboratories TM ); mouse monoclonal anti-ATP5B [clone 3D5AB1] (A21351, 1:1000) (Molecular Probes/Life Technologies Europe BV, Espoo, Finland); rabbit polyclonal anti-DBH (NBP1-31386, 1:1500) (Novus Biologicals, Cambridge, UK); mouse monoclonal anti-Myc [9E10] (ab32, 1:1500) and mouse monoclonal anti-β-actin [AC15] (A1978, 1:2000) (Sigma-Aldrich Finland Oy).

Techniques:

Journal: Journal of inherited metabolic disease

Article Title: Cln3 -mutations underlying juvenile NCL cause significantly reduced levels of Ppt1-protein and Ppt1-enzyme activity in the lysosome

doi: 10.1002/jimd.12106

Figure Lengend Snippet: A. Confocal imaging showing colocalization of CI-M6PR fluorescence with that of Golgi marker, GM-130 [Control (n=30), Patient#1 (n=30) and Patient#2 (n=30)] *p<0.05. Note significantly increased colocalization suggesting impaired exit of CI-M6PR from the TGN in Cln3−/− mouse brain; B.Confocal imaging to show significantly reduced colocalization of Ppt1 and LAMP2 (lysosomal marker) immunofluorescence in cultured cells from JNCL fibroblasts compared with normal (N) fibroblasts. [Control (n=30), Patient#1 (n=30) and Patient#2 (n=30)] *p<0.05. Both Fig. 2A and 2B lower panel are representative image of fibroblast from patient 1. C. Western blot analysis of Ppt1-protein levels in brain lysosomes of 4-, 8-, and 16-mont h old WT and Cln3-mutant mice (n=4); *p<0.05; D. Lysosomal Ppt1-enzyme activity in 4-, 8-, and 16-month old WT and Cln3-mutant mice (n=6); *p<0.05; E. Ppt1 enzyme activity in cultured cells from normal (N) and JNCL patient (P). (n=3); *p<0.05.

Article Snippet: The primary antibodies used for the immunoblots were as follows: Ppt1 (Hofmann Lab; dilution 1:1000) and LAMP2 (EMD Millipore, Cat#MABC40; dilution 1:1000).

Techniques: Imaging, Fluorescence, Marker, Immunofluorescence, Cell Culture, Western Blot, Mutagenesis, Activity Assay

Journal: Journal of inherited metabolic disease

Article Title: Cln3 -mutations underlying juvenile NCL cause significantly reduced levels of Ppt1-protein and Ppt1-enzyme activity in the lysosome

doi: 10.1002/jimd.12106

Figure Lengend Snippet: A. Compared to WT controls, age-dependent progressive increase of autofluorescence in the brain cortex of 4-, 8- and 16-month old Cln3-mutant mice. B. Accumulation of S-palmitoylated proteins in the lysosomes of 4-, 8- and 16-M old WT and Cln3-mutant mice (n=3). C. Qualitative determination of lysosomal pH in the normal (N) and JNCL patient (P) fibroblasts. D. Quantitative determination of lysosomal pH in normal and JNCL patient fibroblasts. Values expressed as the mean ± SD of three different experiments (n=3, *p<0.05). E. Confocal imaging showing colocalization of V0a1 fluorescence with that of lysosomal marker, LAMP2. Note significantly decreased colocalization of PPT1-signal with LAMP2 suggesting lysosomal PPT1 insufficiency in fibroblasts from JNCL patient [Control (n=30), Patient#1 (n=30) and Patient#2 (n=30)] *p<0.05. Fig. 3E lower panel is a representative image of fibroblast from patient 1. F. Confocal imaging showing colocalization of V0a1 fluorescence with that of membrane marker, Na+K+ATPase. Note significantly increased colocalization suggesting the misrouting of V0a1 to the cell membrane of JNCL patient fibroblasts compared with normal fibroblasts (N) [Control (n=30), Patient#1 (n=30) and Patient#2 (n=30)] *p<0.05. Fig. 3F lower panel is representative image of fibroblast from patient 1.

Article Snippet: The primary antibodies used for the immunoblots were as follows: Ppt1 (Hofmann Lab; dilution 1:1000) and LAMP2 (EMD Millipore, Cat#MABC40; dilution 1:1000).

Techniques: Mutagenesis, Imaging, Fluorescence, Marker