Journal: Hepatology Communications

Article Title: Proteomics‐based identification of the role of osteosarcoma amplified‐9 in hepatocellular carcinoma recurrence

doi: 10.1002/hep4.1952

Figure Lengend Snippet: Differential proteins expression in OS‐9 overexpression group (n = 3) and vector control (n = 3) of SMMC‐7721 cells. (A) Volcano plot of the differentially expressed proteins. Gray dots represent genes that are not differentially expressed in the early recurrence group and nonrecurrence group; red dots and blue dots represent genes that are up‐regulated and down‐regulated significantly in the early recurrence group. (B) Heat map of the differentially expressed proteins. Red rectangles mean that genes are up‐regulated in these samples, and blue ones mean down‐regulated. Two hundred and sixty‐eight protein expressions were up‐regulated and 140 protein expressions were down‐regulated in the overexpression group compared with the vector control group (fold change ≥ 1.5; p < 0.05). (C) Heat map of the differentially expressed proteins classified to the hypoxia‐inducible factor 1 (HIF‐1) and tumor necrosis factor (TNF) signaling pathway. (D) Ridgeline plot of Kyoto Encyclopedia of Genes and Genomes pathway enrichment for differentially expressed proteins. (E) Gene Ontology functions for differentially expressed proteins. The left side of the circle includes all related genes, and the right side displays the Gene Ontology terms. Red and blue rectangles mean that genes are up‐regulated and down‐regulated in the early recurrence group. Abbreviations: ALDOA, aldolase, fructose‐bisphosphate A; ALDOC, aldolase, fructose‐bisphosphate C; BCL10, BCL10 immune signaling adaptor; CASP7, caspase 7; CCNB1, cyclin B1; CDK6, cyclin dependent kinase 6; CEBPB, CCAAT enhancer binding protein beta; CHEK2, checkpoint kinase 2; CREBBP, CREB binding protein; DDX58, DExD/H‐box helicase 58; ENO1, enolase 1; ENO2, enolase 2; ENO3, enolase 3; HK2, hexokinase 2; HMOX1, heme oxygenase 1; IGFBP3, insulin like growth factor binding protein 3; JUNB, JunB proto‐oncogene; LDHA, lactate dehydrogenase A; MALT1, MALT1 paracaspase; MLKL, mixed lineage kinase domain like pseudokinase; OE, overexpressed; PGK1, phosphoglycerate kinase 1; PLCG2, phospholipase C gamma 2; SERPINE1, serpin family E member 1; SFN, stratifin. NC, control; STEAP3, STEAP3 metalloreductase; TNFAIP3, TNF alpha induced protein 3; TRAF5, TNF receptor associated factor 5

Article Snippet: Inhibitors including HIF‐1α and tumor necrosis factor alpha (TNF‐α) were obtained (VH‐298, MedChemExpress; HY‐100947, Methylthiouracil; HY‐B0513, MedChemExpress).

Techniques: Expressing, Over Expression, Plasmid Preparation, Control, Binding Assay

Journal: Hepatology Communications

Article Title: Proteomics‐based identification of the role of osteosarcoma amplified‐9 in hepatocellular carcinoma recurrence

doi: 10.1002/hep4.1952

Figure Lengend Snippet: (A–K) The relative messenger RNA (mRNA) expression level of lineage kinase domain‐like MLKL (A), tumor necrosis factor alpha–induced protein 3 (TNFAIP3) (B), JunB proto‐oncogene (JUNB) (C), and tumor necrosis factor receptor–associated factor 5 (TRAF5) (D), TNF‐α (E) and caspase‐7 (CASP7) (F) could be observed to increase more than 2‐fold ( p < 0.01). The relative expression of enolase1 (ENO1) (G), enolase2 (ENO2) (H), enolase3 (ENO3) (I), aldolase, fructose‐bisphosphate A (ALDOA) (J), and lactate dehydrogenase A (LDHA) (K) were also elevated more than 2‐fold ( p < 0.01)

Article Snippet: Inhibitors including HIF‐1α and tumor necrosis factor alpha (TNF‐α) were obtained (VH‐298, MedChemExpress; HY‐100947, Methylthiouracil; HY‐B0513, MedChemExpress).

Techniques: Expressing

Journal: JCI Insight

Article Title: RAGE activation in macrophages and development of experimental diabetic polyneuropathy

doi: 10.1172/jci.insight.160555

Figure Lengend Snippet: ( A ) Kymographs of LysoTracker-labeled organelles in axons from dorsal root ganglia (DRG) neurons with 1.0 U/mL insulin. The horizontal and vertical arrows indicate retrograde direction and recording time (4 minutes), respectively. ( B – E ) The percentage of organelles in 100 μm axon segments that moved anterogradely ( B ), retrogradely ( C ), bidirectionally ( D ), or were stationary ( E ). n = 18–21 axons from 3 independent experiments. ( F ) The velocity of retrograde movements (RV) in 100 μm axon segments. The data consisted of 200–300 movements. ( G ) Kymographs in axons from DRG neurons treated with vehicle and insulin receptor antagonist (BMS-754807, 300 or 500 nmol/L). The stimulation time was 60 minutes. The horizontal and vertical arrows indicate retrograde direction and recording time (4 minutes), respectively. ( H – K ) The percentage of organelles in 100 μm axon segments that moved anterogradely ( H ), retrogradely ( I ), or bidirectionally ( J ), or were stationary ( K ). n = 18–21 axons from 3 independent experiments. ( L ) RV in 100 μm axon segments in each treatment condition. ( M ) Kymographs in axons from DRG neurons treated with vehicle, TNF-α, and TNF-α + JNK inhibitor (SP600125). The stimulation time was 20 minutes. The vertical arrow indicates recording time (4 minutes). ( N – Q ) The percentage of organelles in 100 μm axon segments that moved anterogradely ( N ), retrogradely ( O ), bidirectionally ( P ), or were stationary ( Q ). n = 18–21 axons from 3 independent experiments. ( R ) RV in 100 μm axon segments. The data consisted of 200–300 movements. The data are presented as the mean ± SD. Because the experiments of G – L and M – R were performed contemporaneously, statistical analysis was done using same vehicle control. Statistical analysis was performed by Student’s 2-tailed unpaired t test for B – F and by 1-way ANOVA with Tukey’s multiple-comparison test for H – L and N – R . ** P < 0.01, *** P < 0.001.

Article Snippet: In the experiments with an insulin/IGF-1 receptor antagonist shown in , or with TNF-α with or without a JNK inhibitor shown in , neurons were maintained in DMEM/F12 with 2% B27 and were pretreated with PBS containing 0.1% BSA (vehicle), 300 nmol/L, or 500 nmol/L BMS-754807 (MedChemExpress); 20 ng/mL TNF-α (410-MT, R&D Systems); or 20 ng/mL TNF-α + 50 nmol/L JNK inhibitor (SP600125) (1496, TOCRIS) as indicated in the text.

Techniques: Labeling, Control, Comparison

Journal: The Journal of Experimental Medicine

Article Title: NLRP3 signaling drives macrophage-induced adaptive immune suppression in pancreatic carcinoma

doi: 10.1084/jem.20161707

Figure Lengend Snippet: NLRP3 expression in human and mouse PDA. (A) Lysate from 3-mo-old WT, KC, and KC;NLRP3 −/− mice were tested for expression of IL-1β and IL-18 by Western blotting. Ponceau staining is shown. Experiments were repeated three times. Representative data are shown. (B) Frozen sections of pancreata of mouse PDA tumors were tested for coexpression of CD11b and NLRP3 or CK19 and NLRP3 compared with respective isotype controls. Bar, 10 µm. (C) F4/80 + Gr1 − CD11c − CD11b + macrophages from pancreata or spleen macrophages from 3-mo-old KC mice were tested for expression of NLRP3 compared with isotype controls. (D) Macrophages from pancreata or spleen of KC mice were tested for coexpression of MHC II and CD206. (E) MHC II − CD206 + and MHC II + CD206 − pancreatic macrophage subsets from 3-mo-old KC mice were gated and tested for expression of NLRP3 and IL-1β. Representative and quantitative data are shown. Positive gates are based on isotype controls (not depicted). (F) MHC II − CD206 + and MHC II + CD206 − TAM subsets from WT mice bearing orthotopic PDA were gated and tested for expression of NLRP3 and IL-1β. (G) Macrophages from WT control pancreata or pancreata or spleen of WT mice harboring orthotopic KPC tumors were tested for expression of NLRP3. (H) Paraffin-embedded sections of human PDA were tested for expression of NLRP3 compared with isotype control. Bar, 20 µm. (I) CD15 + monocytic cells from single-cell suspensions of human PDA or PBMCs were gated by flow cytometry and tested for expression of NLRP3. Representative contour plots and quantitative data from six patients are shown. (J) Splenic macrophages from WT mice were cultured alone or in a 5:1 ratio with KPC-derived tumor cells. At 24 h, macrophages were tested for expression of CD206, IL-10, and NLRP3. (K) Similarly, BMDMs from WT mice were stimulated with recombinant TGF-β or TNF and tested for NLRP3 expression. (L) Orthotopic PDA-bearing mice were serially treated with a neutralizing TGF-β mAb or isotype control. Tumors were harvested on day 21, and expression of NLRP3 and CD206 in TAMs was determined by flow cytometry. n = 5/group. All mouse experiments were repeated a minimum of twice using five mice per experimental group. Littermate controls were used. Unpaired Student’s t test was used for statistical analyses. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Data are presented as mean ± standard error. MΦ, macrophage; Ms, mouse; Panc, pancreas; PanIN, pancreatic intraepithelial neoplasia; SSA, side scatter.

Article Snippet: In some experiments, day-10 BMDMs were treated with 8 pg/ml of recombinant mouse TNF (Cell Signaling Technology) or 0.2 ng/ml TGF-β (R&D Systems) for 18 h before analysis for NLRP3 expression by flow cytometry.

Techniques: Expressing, Western Blot, Staining, Control, Flow Cytometry, Cell Culture, Derivative Assay, Recombinant

Journal: Advanced Science

Article Title: Metabolic Reprogramming of Macrophages by Biomimetic Melatonin‐Loaded Liposomes Effectively Attenuates Acute Gouty Arthritis in a Mouse Model

doi: 10.1002/advs.202410107

Figure Lengend Snippet: The targeting capability of MLT‐MLP. A) Uptake of DiI‐labeled MLT‐LP and MLT‐MLP (red) by RAW264.7 cells (blue). Scale bar, 100 µm. B) Fluorescence intensity quantitation of nanoparticles (MLT‐LP, MLT‐MLP) corresponding to (A). C) Uptake of different DiI‐labeled nanoparticles by HUVECs with or without TNF‐α stimulation. Scale bar, 100 µm. D) Fluorescence intensity quantitation of different nanoparticles (MLT‐LP, MLT‐MLP and Blocked MLT‐MLP) corresponding to (C). E) Flow cytometry analysis of ICAM‐1 and VCAM‐1 on HUVECs with or without TNF‐α stimulation. F) Quantification of the mean fluorescence intensity of the flow cytometry results corresponding to (E). G) In vivo fluorescence imaging of gout mice after intravenous injection of free DiR, DiR‐labeled MLT‐LP and DiR‐labeled MLT‐MLP. H) Distribution of MLT‐MLP in various organs compared with free DiR and DiR‐labeled MLT‐LP. I) Fluorescence intensity quantitation results corresponding to (H). J) Fluorescence images and intensity of MLT‐MLP (red), ICAM‐1, and VCAM‐1 (green) in normal or gout paws. In all experiments, data are presented as mean ± SD for n = 3 biological replicates. * p < 0.05, *** p < 0.001; ns, not significant.

Article Snippet: Recombinant mouse TNF‐α were purchased from Novoprotein Biotechnology (Shanghai, China).

Techniques: Labeling, Fluorescence, Quantitation Assay, Flow Cytometry, In Vivo, Imaging, Injection

Journal: Advanced Science

Article Title: Metabolic Reprogramming of Macrophages by Biomimetic Melatonin‐Loaded Liposomes Effectively Attenuates Acute Gouty Arthritis in a Mouse Model

doi: 10.1002/advs.202410107

Figure Lengend Snippet: Anti‐inflammatory effect of MLT‐MLP in vitro. A) Uptake of DiI‐labeled MLT‐MLP (red) by iBMDMs (blue). Scale bar, 100 µm. B) Fluorescence imaging of LPS+MSU stimulated iBMDMs stained with the DCFH‐DA probe (green) and quantitative analysis of ROS levels under different treatment conditions. Scale bar, 200 µm. C, D) Quantitative analysis of intracellular ROS levels in iBMDMs using flow cytometry. E) Detection of inflammatory cytokines (TNF‐α, IL‐6, and IL‐1β) produced from BMDMs stimulated with LPS+MSU or not, with different treatments. F) Immunoblot analysis of proteins from the supernatant (SN) and whole cell lysates (WCL) of BMDMs stimulated with LPS+MSU or not, with different treatments.G) Western blot analysis of ASC oligomerization in BMDMs with different treatments. In all experiments, data are presented as mean ± SD for n = 3 biological replicates. ** p < 0.01, *** p < 0.001; ns, not significant.

Article Snippet: Recombinant mouse TNF‐α were purchased from Novoprotein Biotechnology (Shanghai, China).

Techniques: In Vitro, Labeling, Fluorescence, Imaging, Staining, Flow Cytometry, Produced, Western Blot

Journal: Advanced Science

Article Title: Metabolic Reprogramming of Macrophages by Biomimetic Melatonin‐Loaded Liposomes Effectively Attenuates Acute Gouty Arthritis in a Mouse Model

doi: 10.1002/advs.202410107

Figure Lengend Snippet: MLT‐MLP regulated the metabolic patterns of inflammatory macrophages stimulated with LPS+MSU through mTOR pathway. A) The scheme of metabolic pattern in M1 and M2 type macrophages. B) Detection of extracellular glucose levels of iBMDMs with different treatments ( n = 3). C) Detection of extracellular lactic acid levels of iBMDMs with different treatments ( n = 3). D) Detection of relative ATP production levels of iBMDMs with different treatments ( n = 3). E) Representative ECAR profiles and corresponding parameter analysis of iBMDMs with different treatments ( n = 5). F) Representative OCR profiles and corresponding parameter analysis of iBMDMs with different treatments ( n = 5). G) Correlation analysis of relative lactic acid and ATP levels with iBMDMs polarization parameters levels, including iNOS and Arg‐1. H) Correlation analysis of relative lactic acid and ATP levels with inflammatory cytokines levels, including TNF‐α and IL‐6. I) Representative immunoblots and densitometric analysis for mTOR and p‐mTOR, S6, and p‐S6 in BMDMs stimulated with LPS+MSU or not, with different treatments. In all experiments, data are presented as mean ± SD. ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant mouse TNF‐α were purchased from Novoprotein Biotechnology (Shanghai, China).

Techniques: Western Blot

Journal: Advanced Science

Article Title: Metabolic Reprogramming of Macrophages by Biomimetic Melatonin‐Loaded Liposomes Effectively Attenuates Acute Gouty Arthritis in a Mouse Model

doi: 10.1002/advs.202410107

Figure Lengend Snippet: Treatment effect of MLT‐MLP in vivo. A) The experimental scheme. B) Change in paw swelling of gout mice under different treatments. C) Representative photographs of swelling paw obtained at 8 h after MSU injection. D) Photomicrographs stained by H&E. Scale bar, 100 µm. E) Immunohistochemical images of paw tissues in different groups. Scale bar, 100 µm. F,G) Immunofluorescence staining of Ly6G in paw tissues from different groups and corresponding fluorescence intensity quantitation. Scale bar, 100 µm. H–J) Detection of inflammatory cytokines (TNF‐α, IL‐6, and IL‐1β) in the serum of gout mice with different treatments. In all experiments, data are presented as mean ± SD for n = 5 biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant.

Article Snippet: Recombinant mouse TNF‐α were purchased from Novoprotein Biotechnology (Shanghai, China).

Techniques: In Vivo, Injection, Staining, Immunohistochemical staining, Immunofluorescence, Fluorescence, Quantitation Assay