Journal: JBMR Plus

Article Title: USP34 attenuates cartilage degradation in temporomandibular joint osteoarthritis by ANT1-mediated mitophagy

doi: 10.1093/jbmrpl/ziag004

Figure Lengend Snippet: Deletion of Usp34 accelerates cartilage destruction during TMJ OA. (A) Representative images of Safranin O/Fast Green staining of SHAM and UBR-induced TMJ OA mice. Scale bars: 100 μm for low and 50 μm for high magnification. (B and C) Quantitative analysis regarding cartilage thickness and modified Mankin score according to Safranin O/Fast Green staining. n = 6 per group. (D) Representative micro-CT images reveal the subchondral bone microstructure. Scale bars: 100 μm. (E) Quantitative analysis of subchondral bone parameters. (F and G) Representative images and quantitative analysis of western blot for MMP13, ADAMTS5, α-tubulin in ATDC5 cells transfected with Usp34 siRNA following exposure to IL-1β. (H) Relative mRNA expression of MMP3, MMP13, ADAMTS4, and ADAMTS5 in ATDC5 cells transfected with Usp34 siRNA following exposure to IL-1β.

Article Snippet: To model the inflammatory microenvironment of TMJ OA, ATDC5 cells (1 × 10 5 /well) were cultured in 24-well plates, followed by treatment with 10 ng/mL IL-1β (HY-P7073A; MedChemExpress).

Techniques: Staining, Modification, Micro-CT, Western Blot, Transfection, Expressing

Journal: JBMR Plus

Article Title: USP34 attenuates cartilage degradation in temporomandibular joint osteoarthritis by ANT1-mediated mitophagy

doi: 10.1093/jbmrpl/ziag004

Figure Lengend Snippet: USP34 deubiquitinates and stabilizes ANT1. (A) Volcano plots showing the differentially expressed proteins of USP34-deficient cells from public proteomic dataset in the National Genomics Data Center under accession numbers: OMIX007639. (B) Heatmap showing the differentially expressed protein of USP34-deficient cells from public proteomic dataset (OMIX007639). (C and D) Representative images and quantitative analysis of western blot for ANT1 and α-tubulin in ATDC5 cells transfected with Usp34 siRNA following exposure to IL-1β. (E) Representative images of immunofluorescence staining of ANT1 in the TMJ cartilages of SHAM and UBR-induced TMJ OA mice. Scale bars: 50 μm. (F) Co-immunoprecipitation of USP34 with ectopically expressed ANT1 in HEK293T cells. (G) Immunoblot of ANT1-linked polyubiquitin. HEK293T cells were treated with 10 μM MG132 for 4 h after transfection with the indicated constructs. The cell lysates were subjected to immunoprecipitation with the indicated antibody. (H) Measurement of ANT1 degradation rate. HEK293T cells were transfected with the indicated constructs and treated with 10 mg/mL CHX.

Article Snippet: To model the inflammatory microenvironment of TMJ OA, ATDC5 cells (1 × 10 5 /well) were cultured in 24-well plates, followed by treatment with 10 ng/mL IL-1β (HY-P7073A; MedChemExpress).

Techniques: Western Blot, Transfection, Immunofluorescence, Staining, Immunoprecipitation, Construct

Journal: JBMR Plus

Article Title: USP34 attenuates cartilage degradation in temporomandibular joint osteoarthritis by ANT1-mediated mitophagy

doi: 10.1093/jbmrpl/ziag004

Figure Lengend Snippet: ANT1 overexpression rescues mitochondrial homeostasis in USP34-deficient cells. (A and B) Representative images and quantitative analysis of western blot for LC3, Parkin, PINK1, and α-tubulin in ATDC5 cells transfected with Usp34 siRNA or Lv-ANT1 , following exposure to IL-1β. (C) Representative TEM images of ATDC5 cells transfected with Usp34 siRNA or Lv-ANT1 . Subcellular structures with discernible mitochondria (yellow arrows) and bound by a double limiting membrane are identified as putative mitophagosome structures (red arrows). Scale bars: 200 nm. (D) ATDC5 cells stained with mitotracker and lysotracker after transfection with Usp34 siRNA or Lv-ANT1 . Scale bars: 20 μm. (E) ATDC5 cells stained with Mito-SOX after transfection with Usp34 siRNA or Lv-ANT1 . Scale bars: 20 μm.

Article Snippet: To model the inflammatory microenvironment of TMJ OA, ATDC5 cells (1 × 10 5 /well) were cultured in 24-well plates, followed by treatment with 10 ng/mL IL-1β (HY-P7073A; MedChemExpress).

Techniques: Over Expression, Western Blot, Transfection, Membrane, Staining

Journal: JBMR Plus

Article Title: USP34 attenuates cartilage degradation in temporomandibular joint osteoarthritis by ANT1-mediated mitophagy

doi: 10.1093/jbmrpl/ziag004

Figure Lengend Snippet: USP34 overexpression enhanced chondrocyte viability. (A and B) Representative images and quantitative analysis of western blot for LC3, Parkin, PINK1, and α-tubulin in ATDC5 cells transfected with Usp34 lentiviral activation particles ( Usp34 ac) following exposure to IL-1β. (C and D) Representative images and quantitative analysis of western blot for MMP13, ADAMTS5, and α-tubulin in ATDC5 cells transfected with Usp34 ac following exposure to IL-1β. (E and F) Representative images and quantitative analysis of western blot for MMP13, ADAMTS5, and α-tubulin in ATDC5 cells with the indicated treatment. (G) ATDC5 cells stained with Acan and Col2a1 after transfection with Usp34 ac or Lv-ANT1 . Scale bars: 50 μm. (H and I) Relative mRNA expression of Acan and Col2a1 in ATDC5 cells with the indicated treatments.

Article Snippet: To model the inflammatory microenvironment of TMJ OA, ATDC5 cells (1 × 10 5 /well) were cultured in 24-well plates, followed by treatment with 10 ng/mL IL-1β (HY-P7073A; MedChemExpress).

Techniques: Over Expression, Western Blot, Transfection, Activation Assay, Staining, Expressing

Journal: International Journal of Cardiology. Cardiovascular Risk and Prevention

Article Title: The sphingolipid metabolite sphingosine protects against hypertension by targeting metabolic-inflammatory crosstalk via the NLRP3 inflammasome

doi: 10.1016/j.ijcrp.2025.200562

Figure Lengend Snippet: SPH attenuates pathological changes and suppresses NLRP3 inflammasome activation in the hearts of hypertensive mice. (A, B) Masson's trichrome staining of heart (A) and aorta (B) sections, showing reduced collagen deposition (blue) with SPH treatment. Scale bars: 20 μm for heart, 50 μm (main) and 20 μm (inset) for aorta. (C) H&E staining of heart sections showing amelioration of myocardial disarray and inflammation. Scale bars: 500 μm (main) and 20 μm (inset).(D) ELISA quantification of serum IL-18 and IL-1β levels. Data are mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 vs. HBP group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Serum levels of interleukin-18 (IL-18) and interleukin-1β (IL-1β) were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits (IL-18: Lianke, Cat#EK218-96; IL-1β: Lianke, Cat# EK201BHS-96) according to the manufacturers' instructions.

Techniques: Activation Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Cardiology. Cardiovascular Risk and Prevention

Article Title: The sphingolipid metabolite sphingosine protects against hypertension by targeting metabolic-inflammatory crosstalk via the NLRP3 inflammasome

doi: 10.1016/j.ijcrp.2025.200562

Figure Lengend Snippet: SPH mitigates Angiotensin II-induced inflammasome activation, apoptosis, and oxidative stress in HUVECs. (A, B) Representative immunofluorescence images showing increased expression of NLRP3 (red, A) and ASC (green, B) in AngII-treated cells, which is reduced by subsequent SPH treatment. Scale bar = 100 μm. (C) TUNEL assay (green) demonstrating increased apoptosis in AngII-treated cells, which is attenuated by SPH. Scale bar = 100 μm. (D, E) Biochemical assays showing that SPH or MCC950 treatment reverses the AngII-induced decrease in SOD1 activity (G).NO (H) and increase in MDA levels (D). (F, G) DCFH-DA staining showing increased intracellular ROS (green) after AngII treatment, which is suppressed by SPH or MCC950. Representative images (F) and quantification (E) are shown. (I, J) ELISA results showing that SPH or MCC950 treatment inhibits the AngII-induced secretion of IL-1β (I) and IL-18 (J). (K) Scanning electron microscopy (SEM) images showing that SPH or MCC950 treatment improves the cell surface morphology and reduces features of pyroptotic damage induced by AngII. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Serum levels of interleukin-18 (IL-18) and interleukin-1β (IL-1β) were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits (IL-18: Lianke, Cat#EK218-96; IL-1β: Lianke, Cat# EK201BHS-96) according to the manufacturers' instructions.

Techniques: Activation Assay, Immunofluorescence, Expressing, TUNEL Assay, Activity Assay, Staining, Enzyme-linked Immunosorbent Assay, Electron Microscopy

Journal: Materials Today Bio

Article Title: Targeting neuroinflammation and PVN CRH neurons: dexmedetomidine liposomes for perioperative neurocognitive disorder with comorbid anxiety

doi: 10.1016/j.mtbio.2025.102634

Figure Lengend Snippet: D@ACLipo alleviates surgery-induced microglia activation and neuroinflammation. (A) Schematic illustration of the signaling pathway for D@ACLipo to alleviate surgery-induced microglia activation and neuroinflammation. (B) Representative fluorescence images of microglia (green) taking up ICG-D@CLipo (red) and ICG-D@ACLipo (red) in the hippocampal CA1 region. Scale bar: 20 μm. (C–F) The protein levels of TREM2, TLR4, p-P65, and P65 were assessed by western blot. (G) Representative fluorescence images of microglia (green) and the images from Sholl analysis in the CA1 region. Scale bar: 20 μm. Average soma size (H) and relative fluorescence intensity (I) of microglia. (J) The number of intersections from the soma center in Sholl analysis of hippocampal microglia. The relative mRNA expression of Arg1 (K), and iNOS (L) were measured by qRT-PCR. The concentrations of IL-6 (M), IL-1β (N), and TNF-α (O) in the hippocampus were measured by ELISA. Data are shown as mean ± SEM and analyzed by one-way ANOVA with Tukey's post hoc test. n = 5. ns, no significant difference, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: According to the manufacturer's instructions, the concentrations of IL-6, IL-1β, and TNF-α in the hippocampus, and cortisol in serum were measured by using ELISA kits (CSB-E04639m, CSB-E08054m, CSB-E04741m, and CSB-E05113m, respectively; CUSABIO).

Techniques: Activation Assay, Fluorescence, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Materials Today Bio

Article Title: Targeting neuroinflammation and PVN CRH neurons: dexmedetomidine liposomes for perioperative neurocognitive disorder with comorbid anxiety

doi: 10.1016/j.mtbio.2025.102634

Figure Lengend Snippet: D@ACLipo alleviates surgery-induced PVN CRH neurons hyperactivation. (A) Schematic illustration of the mechanism for D@ACLipo alleviate surgery-induced anxiety-like behaviors. (B) Schematic diagram showing the timeline of the experimental procedure. (C) Representative fluorescence images of c-Fos (green) and co-immunostained c-Fos (green) and CRH (red) in the PVN region. Scale bar: 200 μm (up) and 50 μm (down). (D) Statistical results of c-Fos-positive cells in the PVN region from the indicated groups. (E) Quantification of the percentage of c-Fos-positive neurons expressing CRH in the PVN region from the indicated groups. (F) Quantification of the percentage of CRH-positive neurons expressing c-Fos in the PVN region from the indicated groups. (G) Serum cortisol concentration detected by ELISA. (H) Schematic diagram of optical fiber photometry in mice. (I) Representative images of GCaMp6m viral expression in PVN CRH neurons. Scale bar: 100 μm (left) and 20 μm (right). (J) Representative images showing changes in GCaMP6m signals in PVN CRH neurons (gray lines represent individual mouse signals, red lines represent means, and red shadings represent SDs). (K) Quantification of the changes in GCaMP6m signals from the indicated groups. Data are shown as mean ± SEM and analyzed by one-way ANOVA with Tukey's post hoc test. n = 5. ns, no significant difference, ∗ P < 0.05, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: According to the manufacturer's instructions, the concentrations of IL-6, IL-1β, and TNF-α in the hippocampus, and cortisol in serum were measured by using ELISA kits (CSB-E04639m, CSB-E08054m, CSB-E04741m, and CSB-E05113m, respectively; CUSABIO).

Techniques: Fluorescence, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Cancer Communications

Article Title: The Ly6g high Neutrophil Subset Dictates Breast Cancer Lung Metastasis via CD8 + T Cell Death

doi: 10.34133/cancomm.0003

Figure Lengend Snippet: Il-1β induces Ly6g high neutrophil NETosis in the lung metastatic niche. (A) Heatmap of the scRNA-seq data showing the expression of cytokine genes at different time points during lung metastasis. (B and C) Representative immunofluorescence micrographs (B) showing NET formation by FACS-sorted Ly6g high and Ly6g low neutrophils ( n = 6) after treatment with Il-1β, Cxcl2, and Ccl6 for 6 h in vitro. NETs were stained with antibodies against Mpo (red) and H3cit (green), and nuclei were counterstained with DAPI (blue). The statistical data are presented in (C). (D) Representative immunofluorescence micrographs showing NET formation at the MACRO stages of lung tissue with PBS, rIl-1β, anti-IgG, and anti-Il-1β antibody treatment, respectively [4T1-LM3 (BALB/c) model, n = 5]. NETs were stained with antibodies against Mpo (red) and H3cit (green), and nuclei were counterstained with DAPI (blue). The bar graph on the right quantifies NET formation. (E) Representative bioluminescence imaging and hematoxylin and eosin (H&E) staining images at the MACRO lungs from mice treated with PBS, rIl-1β, IgG, or anti-Il-1β antibody [4T1-LM3 (BALB/c) model, n = 5]. The bar graph on the right shows the quantitative data of lung metastasis burden. (F) Violin plots showing the expression of Il1b in different cell clusters in the lung tissues based on scRNA-seq data from Fig. D. (G) Representative immunofluorescence micrographs demonstrate NET formation in sorted Ly6g high neutrophils ( n = 6). Neutrophils were treated with CM-MΦ or CM-MΦ that had been neutralized with an anti-Il-1β antibody. NETs were stained with antibodies against Mpo (red) and H3cit (green), and nuclei were counterstained with DAPI (blue). (H and I) Mice were treated with anti-IgG control, anti-F4/80 antibody, or anti-F4/80 antibody combined with rIl-1β until the macrometastatic stage [4T1-LM3 (BALB/c) model, n = 6]. (H) Il-1β levels in the lungs were detected by ELISA. (I) Representative immunofluorescence images show NET formation. NETs were stained for Mpo (red) and H3cit (green), and nuclei were counterstained with DAPI (blue). The bar graph on the right quantifies NET formation (I). (J) Macrophages were treated with CM-Neu, NETs (5 μg/ml), NETs (10 μg/ml), or NETs (10 μg/ml) combined with deoxyribonuclease (DNase) I ( n = 3). The expression of Il1b was determined by qPCR. The data with error bars are presented as the mean ± SD; statistical significance was determined by 2-way ANOVA (C) and 1-way ANOVA test (D, E, and G to J). 4T1-LM3, 4T1-lung metastasis 3; ANOVA, analysis of variance; Ccl11 , c-c motif chemokine ligand 11; Ccl12 , c-c motif chemokine ligand 12; Ccl17 , c-c motif chemokine ligand 17; Ccl2 , c-c motif chemokine ligand 2; Ccl22 , c-c motif chemokine ligand 22; Ccl3 , c-c motif chemokine ligand 3; Ccl4 , c-c motif chemokine ligand 4; Ccl5 , c-c motif chemokine ligand 5; Ccl6 , c-c motif chemokine ligand 6; CCL6; c-c motif ligand 6; Ccl9 , c-c motif chemokine ligand 9; CM-MΦ, macrophage-derived conditioned medium; CM-Neu, neutrophil-derived conditioned medium; Cxcl12 , c-x-c motif chemokine ligand 12; Cxcl14 , c-x-c motif chemokine ligand 14; Cxcl16 , c-x-c motif chemokine ligand 16; CXCL2, c-x-c motif chemokine ligand 2; Cxcl2 , c-x-c motif chemokine ligand 2; Cxcl3 , c-x-c motif chemokine ligand 3; Cxcl9 , c-x-c motif chemokine ligand 9; DAPI, 4’,6-diamidino-2-phenylindole; ELISA, enzyme linked immunosorbent assay; FACS, fluorescence-activated cell sorting; H3cit; citrullinated histone H3; Il12a , interleukin 12a; Il13 , interleukin 13; Il18 , interleukin, 18; Il1a , interleukin 1α; Il1b , interleukin 1β; Il-1β, interleukin-1β; Il2 ,interleukin 2; Il33 , interleukin 33; Il4 , interleukin 4; Il6 , interleukin 6; Ly6g, lymphocyte antigen 6 complex locus g; MACRO, macrometastatic lung; MICRO, micrometastatic lung; MPO, myeloperoxidase; NETs, neutrophil extracellular trap; NK, natural killer; NL, normal lung; Ppbp , pro-platelet basic protein; Neu, neutrophil; PRE, premetastatic lung; qRT-PCR, quantitative real-time polymerase chain reaction; rIl-1β, recombinant interleukin-1β; scRNA-seq: single-cell RNA sequencing; SD, standard deviation.

Article Snippet: To induce NET formation, primary tumors were resected on day 21 after the orthotopic injection of 4T1-LM3 cells, followed by daily intraperitoneal injections of recombinant Il-1β (rIl-1β; 8 ng per mouse, HY-P7073, MedChemExpress) until the macrometastatic stage.

Techniques: Expressing, Immunofluorescence, In Vitro, Staining, Imaging, Control, Enzyme-linked Immunosorbent Assay, Derivative Assay, Fluorescence, FACS, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Recombinant, RNA Sequencing, Standard Deviation

Journal: Cancer Communications

Article Title: The Ly6g high Neutrophil Subset Dictates Breast Cancer Lung Metastasis via CD8 + T Cell Death

doi: 10.34133/cancomm.0003

Figure Lengend Snippet: Prognostic significance of NETs in human BC. (A) Representative FACS plot showing the ratio of human CD84 high and CD84 low neutrophils in healthy individuals ( n = 50) and patients with BC at different stages [stages I/II ( n = 80), stages III/IV ( n = 80)]. To define the CD84 high and CD84 low subsets in humans, we first established the positive gating threshold using FMO controls. Subsequently, the boundary between “high” and “low” subsets was determined based on a clear inflection point observed in the fluorescence intensity histogram. Statistical significance was determined by comparing with the healthy group. The bar graph on the right quantifies the ratio of human CD84 high and CD84 low neutrophils. (B) Representative immunofluorescence micrographs showing NET formation of CD84 high and CD84 low neutrophils, which were sorted by FACS after treatment with PMA for 2 h ( n = 6). NETs were stained with antibodies against MPO (red) and H3cit (green), and nuclei were counterstained with DAPI (blue). The bar graph on the right quantifies the formation of NETs. (C) Plasma NET levels in healthy individuals ( n = 50) and BC patients at different stages [stages I/II ( n = 80), stages III/IV ( n = 80)]. (D) Kaplan–Meier survival curves showing the overall survival (OS) of BC patients with low (NETs < 344.91 pg/ml; n = 83) or high (NETs ≥ 344.91 pg/ml; n = 77) concentrations of plasma NETs. BC patients were stratified into high and low NET groups using the mean plasma NET level of the entire cohort as the cutoff. (E) Receiver operator characteristic (ROC) curve analysis of plasma NET levels for predicting BC patients’ lung metastases ( n = 160). The area under the curve (AUC) value reflects the model’s power to distinguish between BC patients with and without lung metastasis within 6 years after diagnosis. Higher AUC values (approaching 1) denote superior differentiation accuracy at this time point. (F) Correlation between plasma NET levels and CD8 + T cell proportion in healthy individuals and patients with BC ( n = 210). (G) Kaplan–Meier analysis showing the recurrence-free survival of BC patients with high or low levels of LL37 ( n = 4,929). Data were obtained from the Kaplan–Meier plotter database, which does not provide detailed numerical thresholds for LL37 level classification. (H) Mechanism scheme of Ly6g high and Ly6g low neutrophils in promoting pulmonary metastasis of BC. Briefly, Ly6g high neutrophils accumulated in the premetastatic stage and induced CD8 + T cell apoptosis through NETosis. The NET-derived cathelicidin directly bound with Ant1, an mPTP protein in CD8 + T cells, leading to conformational changes in the Ant1 and subsequent Ant1–Vdac1 complex formation, which resulted in mPTP opening, loss of ΔΨm, and uncoupling of mitochondrial electron transport chain in CD8 + T cells. Ly6g low neutrophils bearing MDSC-like transcriptional signatures exhibit a superior capacity to inhibit the proliferation and effector functions of CD8 + T cells. The data with error bars are presented as the mean ± SD; statistical significance was determined by 2-way ANOVA (A), Student’s t test (B), 1-way ANOVA test (C), and 2-sided log-rank test (D and G). 4T1-LM3, 4T1-lung metastasis 3; ANOVA, analysis of variance; APC, allophycocyanin; BC, breast cancer; CD8, cluster of differentiation 8; CD84, cluster of differentiation 84; CI, confidence interval; DAPI, 4',6-diamidino-2-phenylindole; E0771-LM3, E0771-lung metastasis 3; FACS, fluorescence-activated cell sorting; FMO, fluorescence-minus-one; H3cit, citrullinated histone H3; HR, hazard ratio; Interferon-γ, IFN-γ; Il-1β, interleukin-1β; Ly6g, lymphocyte antigen 6 complex locus g; MDSC, myeloid-derived suppressor cell; MPO, myeloperoxidase; mPTP, mitochondrial permeability transition pore; NETs, neutrophil extracellular traps; PADI4, peptidyl arginine deiminase 4; PE, phycoerythrin; PMA, phorbol 12-myristate 13-acetate; RFS, recurrence-free survival; ROS, reactive oxygen species; Vdac1, voltage-dependent anion channel 1; SD, standard deviation; ΔΨm, mitochondrial membrane potential.

Article Snippet: To induce NET formation, primary tumors were resected on day 21 after the orthotopic injection of 4T1-LM3 cells, followed by daily intraperitoneal injections of recombinant Il-1β (rIl-1β; 8 ng per mouse, HY-P7073, MedChemExpress) until the macrometastatic stage.

Techniques: Fluorescence, Immunofluorescence, Staining, Clinical Proteomics, Biomarker Discovery, Derivative Assay, FACS, Permeability, Standard Deviation, Membrane