Journal: Nature communications

Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice.

doi: 10.1038/s41467-023-43650-z

Figure Lengend Snippet: Fig. 1 | PPT1 deficiency protects SLE mice from autoantibodies and nephritis. C57BL/6 J (B6), Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa mice were sacrificed at 16 weeks (n = 5 mice for B6; n = 15 mice for Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa for the entire figure). A Spleen image (left), weight (center) and cell numbers (right) are shown. B Serum anti-DNA antibodies were measured by ELISAs. C Serum anti- RNP/Sm antibodies were measured by ELISAs at 16 weeks. D Serum total IgG were measured by ELISAs. E Glomerulus size was calculated from H&E-stained kidney sections (left). Each dot represents an individual glomerulus (n = 40 in B6; n = 44 in Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa). F Representative images from kidney immunofluorescence sections are shown (left). IgG (top) and C3 (bottom) staining

Article Snippet: The following primary antibodies used for Western blotting were all used at a dilution of 1:1000 (unless otherwise indicated): Sigma Anti-Flag (host: Mouse) Catalog #: F1840 Roche Anti-HA (host: Rat) Catalog #: 11867423001 Abcam Calnexin (host: Rabbit) Catalog #: ab213243 Abcam Rab7 (host: Rabbit) Catalog #: ab137029 Abcam Tubulin (host: Mouse) Catalog #: ab78078 (1:5000 dilution) Novus PPT1 (host: Rabbit) Catalog #: NBP2-93840 Novus TLR9 (host: Mouse) Catalog #: NBP2-24729 CST anti-biotin, HRP-linked Catalog #: 7075 S Abmart GM130 (host: Rabbit) Catalog #: T55142 CST EEA1 (host: Rabbit) Catalog #: 2411 Abcamgoat anti-mouse IgGH&L (HRP) Catalog #: ab6789 (1:5000 dilution) Abcam goat anti-rabbit IgG H&L (HRP) Catalog #: ab6721 (1:5000 dilution) Abcam goat anti-rat IgG H&L (HRP) Catalog #: ab97057 (1:5000 dilution)

Techniques: Staining

Journal: Nature communications

Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice.

doi: 10.1038/s41467-023-43650-z

Figure Lengend Snippet: Fig. 2 | PPT1 inhibitor HDSF suppresses IFNα in SLE patients and SLE patho- genesis in mice. A PBMCs of SLE patients were treated with DMSO or HDSF overnight. After CpG A stimulation, IFNα levels was evaluated by ELISAs (n = 17 individuals for DMSO/HDSF). B 8-weeks-old B6.Sle1yaa mice (n = 7 mice per group for the rest of figure) were treated with DMSO or HDSF for 8 weeks before sacrifice at 16 weeks. Spleen image (left), weight (center) and cell numbers (right) are shown. C Serum anti-DNA antibodies were measured by ELISAs at indicated weeks of treatment. D Serum anti-RNP/Sm antibodies were measured by ELISAs at the end of treatment. E Serum total IgG were measured by ELISAs at indicated weeks of treatment. F Glomerulus size at 16 weeks is calculated from H&E-stained kidney sections (left). Each dot represents an individual glomerulus (n = 40 glomeruli per group). G Representative images from kidney immunofluorescence sections are

Article Snippet: The following primary antibodies used for Western blotting were all used at a dilution of 1:1000 (unless otherwise indicated): Sigma Anti-Flag (host: Mouse) Catalog #: F1840 Roche Anti-HA (host: Rat) Catalog #: 11867423001 Abcam Calnexin (host: Rabbit) Catalog #: ab213243 Abcam Rab7 (host: Rabbit) Catalog #: ab137029 Abcam Tubulin (host: Mouse) Catalog #: ab78078 (1:5000 dilution) Novus PPT1 (host: Rabbit) Catalog #: NBP2-93840 Novus TLR9 (host: Mouse) Catalog #: NBP2-24729 CST anti-biotin, HRP-linked Catalog #: 7075 S Abmart GM130 (host: Rabbit) Catalog #: T55142 CST EEA1 (host: Rabbit) Catalog #: 2411 Abcamgoat anti-mouse IgGH&L (HRP) Catalog #: ab6789 (1:5000 dilution) Abcam goat anti-rabbit IgG H&L (HRP) Catalog #: ab6721 (1:5000 dilution) Abcam goat anti-rat IgG H&L (HRP) Catalog #: ab97057 (1:5000 dilution)

Techniques: Staining

Journal: Nature communications

Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice.

doi: 10.1038/s41467-023-43650-z

Figure Lengend Snippet: Fig. 6 | PPT1 depalmitoylates TLR9 in lysosomes. A ABE assays were performed on RAW264.7 cells transduced with mTLR9-HA after 4 h treatment of indicated TLR agonizts. A representative blot (left) and relative mTLR9 S-palmitoylation (right, quantified as the ratio of the mTLR9 output to the calnexin output, n = 3 replicates). B ABE assays were performed on RAW264.7 cells transduced with mTLR7-HA after 4 h treatment of R848. A representative blot (left) and relative mTLR7 S-palmitoylation (right, quantified as the ratio of the mTLR7 output to the calnexin output, n = 3 replicates). C ABE assays were performed on 293 T cells transfected with indicated depalmitoylating enzymes and mTLR9-FLAG (n = 3 replicates).

Article Snippet: The following primary antibodies used for Western blotting were all used at a dilution of 1:1000 (unless otherwise indicated): Sigma Anti-Flag (host: Mouse) Catalog #: F1840 Roche Anti-HA (host: Rat) Catalog #: 11867423001 Abcam Calnexin (host: Rabbit) Catalog #: ab213243 Abcam Rab7 (host: Rabbit) Catalog #: ab137029 Abcam Tubulin (host: Mouse) Catalog #: ab78078 (1:5000 dilution) Novus PPT1 (host: Rabbit) Catalog #: NBP2-93840 Novus TLR9 (host: Mouse) Catalog #: NBP2-24729 CST anti-biotin, HRP-linked Catalog #: 7075 S Abmart GM130 (host: Rabbit) Catalog #: T55142 CST EEA1 (host: Rabbit) Catalog #: 2411 Abcamgoat anti-mouse IgGH&L (HRP) Catalog #: ab6789 (1:5000 dilution) Abcam goat anti-rabbit IgG H&L (HRP) Catalog #: ab6721 (1:5000 dilution) Abcam goat anti-rat IgG H&L (HRP) Catalog #: ab97057 (1:5000 dilution)

Techniques: Transduction, Transfection

Journal: Nature communications

Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice.

doi: 10.1038/s41467-023-43650-z

Figure Lengend Snippet: Fig. 7 | The palmitoylation cycle regulates TLR9 ligand binding. A TNF pro- duction was measured by intracellular staining in CpG B-treated Tlr9-/- RAW264.7 cells transduced with TLR9 Mut2 (n = 4 replicates). B Immunoprecipitation assay with CpG B-biotin were performed on Tlr9-/- RAW264.7 cells transduced with TLR9 WT or Mut2. A representative blot (left) and relative ligand binding (right, calcu- lated as the ratio of CpG B-bound on cleaved mTLR9 to the total mTLR9 in input) are shown (n = 3 replicates). C TNF production was measured by ELISAs in CpG B-treated Zdhhc3+/+ or Zdhhc3-/- RAW264.7 cells transduced with mTLR9-HA (n = 3 replicates). D Immunoprecipitation assays with CpG B-biotin were performed on Zdhhc3+/+ or Zdhhc3-/- RAW264.7 cells transduced with mTLR9-HA (n = 3 replicates). E TNF production was measured by ELISAs in CpG B-treated Ppt1-/- or Ppt1-/-

Article Snippet: The following primary antibodies used for Western blotting were all used at a dilution of 1:1000 (unless otherwise indicated): Sigma Anti-Flag (host: Mouse) Catalog #: F1840 Roche Anti-HA (host: Rat) Catalog #: 11867423001 Abcam Calnexin (host: Rabbit) Catalog #: ab213243 Abcam Rab7 (host: Rabbit) Catalog #: ab137029 Abcam Tubulin (host: Mouse) Catalog #: ab78078 (1:5000 dilution) Novus PPT1 (host: Rabbit) Catalog #: NBP2-93840 Novus TLR9 (host: Mouse) Catalog #: NBP2-24729 CST anti-biotin, HRP-linked Catalog #: 7075 S Abmart GM130 (host: Rabbit) Catalog #: T55142 CST EEA1 (host: Rabbit) Catalog #: 2411 Abcamgoat anti-mouse IgGH&L (HRP) Catalog #: ab6789 (1:5000 dilution) Abcam goat anti-rabbit IgG H&L (HRP) Catalog #: ab6721 (1:5000 dilution) Abcam goat anti-rat IgG H&L (HRP) Catalog #: ab97057 (1:5000 dilution)

Techniques: Ligand Binding Assay, Staining, Transduction, Immunoprecipitation

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 PPT1 conditional KO mice were generated by employing the CRISPR/Cas9 technology to disrupt macrophage PPT1 and purchased from Cyagen Biosciences (Suzhou) Inc.

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 PPT1 conditional KO mice were generated by employing the CRISPR/Cas9 technology to disrupt macrophage PPT1 and purchased from Cyagen Biosciences (Suzhou) Inc.

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 PPT1 conditional KO mice were generated by employing the CRISPR/Cas9 technology to disrupt macrophage PPT1 and purchased from Cyagen Biosciences (Suzhou) Inc.

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 PPT1 conditional KO mice were generated by employing the CRISPR/Cas9 technology to disrupt macrophage PPT1 and purchased from Cyagen Biosciences (Suzhou) Inc.

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 PPT1 conditional KO mice were generated by employing the CRISPR/Cas9 technology to disrupt macrophage PPT1 and purchased from Cyagen Biosciences (Suzhou) Inc.

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 PPT1 conditional KO mice were generated by employing the CRISPR/Cas9 technology to disrupt macrophage PPT1 and purchased from Cyagen Biosciences (Suzhou) Inc.

Techniques: Inhibition, In Vitro, Control

Journal: The Journal of Biological Chemistry

Article Title: Disruption of lysosomal nutrient sensing scaffold contributes to pathogenesis of a fatal neurodegenerative lysosomal storage disease

doi: 10.1016/j.jbc.2024.105641

Figure Lengend Snippet: Reagent resources and list of primers

Article Snippet: Recombinant Human PPT1 , Creative Biomart , Cat#PPT1-367H.

Techniques: Electron Microscopy, Recombinant, Protease Inhibitor, Staining, Isolation, Clinical Proteomics, Membrane, Enzyme-linked Immunosorbent Assay, Activation Assay, Mutagenesis

Journal: Cancer discovery

Article Title: A unified approach to targeting the lysosome’s degradative and growth signaling roles

doi: 10.1158/2159-8290.CD-17-0741

Figure Lengend Snippet: (A) Schematic demonstrating the pulldown strategy. (B) Chemical structure of the DQ661-photoprobe. (C) A375P cells were treated with the DQ661-photoprobe (24 hr, 0 – 100 μM) and lysates were immunoblotted. (D) Graph depicting the mass spectrometry analysis of lysate from pulldown with the DQ661-photoprobe. Conditions analyzed were cells that were treated with the DQ661-photoprobe ± UV and ± 10X concentration of DQ661 competition. (E) Cathepsin activity in A375P cells following treatment with DQ660 or DQ661 (3 hr, 3 μM). (F) PPT1 enzymatic activity in A375P cells following treatment with DQ660 or DQ661 (3 hr, 3 μM). (G) In vitro binding of DQ661 to PPT1. Differential Scanning Calorimetry of 1 mg/mL PPT1 (29.4 μM) in the absence (black) or presence of DQ661 (100 μM, green). Tm: melting temperature. (H) A375P cells were treated with DQ661 (0 – 240 minutes, 3 μM). CD44 palmitoylation measured using the acyl-biotinyl exchange (ABE) assay increases with DQ661 treatment compared with control. Samples not treated with hydroxylamine (-HAM) serve as a negative control. (I) A375P cells were treated with 25 nM PPT1 siRNA or non-targeting (siNT). Cells were transfected overnight in the absence of serum. Upon serum restimulation, cells were collected 3 or 6 hr thereafter and lysate was immunoblotted. (J) A375P cells were treated with DQ661 (1 hr, 3 μM) in the presence or absence of NTBHA (2 mM). Lysate was immunoblotted.

Article Snippet: Purification of PPT1 Full length PPT1 plasmid was obtained from Addgene (Plasmid #25205).

Techniques: Mass Spectrometry, Concentration Assay, Activity Assay, In Vitro, Binding Assay, Negative Control, Transfection

Journal: Cancer discovery

Article Title: A unified approach to targeting the lysosome’s degradative and growth signaling roles

doi: 10.1158/2159-8290.CD-17-0741

Figure Lengend Snippet: (A) Schematic depicting interactions between vATPase/Ragulator/Rag/mTORC1 and Rheb on the lysosome surface. (B) A375P cells were treated with DQ661 (6 hr, 3 μM) and membrane fractions were immunoblotted for vATPase machinery. Densitometry shown below corresponding blot. (C) PLA was performed on A375P cells treated with DQ661 (6 hr, 1 μM) for the p18 (Ragulator) – V1A physical interaction. (D) A375P cells were treated with DQ661 or QN (6 hr, 3 μM) and IF microscopy was performed to detect changes in p18/LAMP2 localization or (E) RagC/LAMP2 localization or (F) mTOR/LAMP2 localization. Below each panel is the respective co-localization analysis. Data are represented as mean ± SD. N = 50 cells per condition (ANOVA with Dunnett’s multiple comparison test). *p<0.0001 versus Untreated group. (G) A375P cells were treated with DQ661 (6 hr, 3 μM) and lysosome fractions were isolated and immunoblotted. (H) A375P cells were treated with DQ661 (3 hr, 3 μM) and PLA was performed. Blue represents DAPI and red fluorescence represents mTOR-RagC interactions. (I) HEK293T cells expressing FLAG-RagB were treated with DQ661 (4 hr, 3 μM) and immunoprecipitation lysates were probed for mTOR. (J) PLA was performed on A375P cells were treated with rapamycin (3 μM), torin-1 (3 μM), baf (100 nM), siramesine (8 μM), or DQ661 (1 μM) for 3 hr. Blue is DAPI and red fluorescence reflects mTOR-Rheb interaction. (K) A375P cells were treated with HDSF (6 hr, 40 μM) and membrane fractions were isolated and immunoblotted. (L) A375P cells were treated with NT or PPT1 siRNA for 24 hours. Membranes were subsequently fractionated and immunoblotted.

Article Snippet: Purification of PPT1 Full length PPT1 plasmid was obtained from Addgene (Plasmid #25205).

Techniques: Microscopy, Isolation, Fluorescence, Expressing, Immunoprecipitation