Journal: bioRxiv

Article Title: Chemokine receptor activity is differentially regulated by membrane cholesterol

doi: 10.64898/2026.07.20.738555

Figure Lengend Snippet: a , Molecular structure of cholesterol, showing the hydroxylation sites in 25-hydroxycholesterol (HCHL) and 7β-HCHL. b , Changes in N-terminally Venus-tagged β–arrestin2 recruitment to C-terminally Rluc3-tagged CXCR4 in HEK293A cells rescued with HPβC preloaded with 25-HCHL and 7β-HCHL. Changes in dose–response recruitment (left), efficacy (middle), and potency (right). c , Effect of 25-HCHL addition on the recruitment of β–arrestin2 to chemokine receptors, for which efficacy (left), and potency (right) were calculated. d , Changes in the efficacy (left), and the potency (right) for N-terminally Venus-tagged mini-Gα s/i coupling to C-terminally Rluc3-tagged chemokine receptors rescued with 25-HCHL. e , Role of 25-HCHL in the constitutive Gα i activation levels of untagged chemokine receptors, calculated as changes in efficacy (left), and potency (right). f , Summarizing scheme of the role of decreased cholesterol and oxysterol addition in chemokine receptor ligand binding and intracellular signal transduction; created with BioRender.com. ( b-e ) The asterisk symbols indicate statistically significant differences (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns denotes non-significant changes) between the indicated groups, determined by one-way ANOVA. N = 3 independent experiments, performed in technical triplicates. All data are shown as mean ± SD.

Article Snippet: Prior to detergent extraction, CXCR4 membranes were treated with 100 μM IT1t dihydrochloride (MedChemExpress, Cat#HY-101458A).

Techniques: Activation Assay, Ligand Binding Assay, Transduction

Journal: Journal of Biomedical Science

Article Title: Aptamer-based inhibition of MNK1 reduces pancreatic ductal adenocarcinoma growth by targeting cancer stem cells

doi: 10.1186/s12929-026-01275-6

Figure Lengend Snippet: apMNKQ2 targets the CSC compartment in PDAC. A, B Left: Representative images of colonies ( A ) or spheres ( B ) in control- or apMNKQ2-transfected Panc354 cells. Right: Mean ± SEM in the crystal violet optical density (OD) ( A ) or number (no.) of spheres/ml ( B ) in control- or apMNKQ2-transfected Panc215 or Panc354 cells. * p < 0.05, **** p < 0.0001; as determined by one-sample t test. C Mean ± SEM of the percentage of CD24-, CD133-, CXCR4- or ALDH-positive cells in control- or apMNKQ2-transfected cells 24 h post transfection. *** p < 0.001, **** p < 0.0001; as determined by one-sample t test. D Schematic of the transfection groups, apMNKQ2-FITC transfection efficiency determined by flow cytometry and subsequent dilution of cells for ELDA determination. E Percentage of tumor take (number of tumors confirmed/number of injections) for subcutaneously injected control (CTL)- and apMNKQ2 (Q2)-transfected cells was determined over the course of 124 days. F Tumor weight (g) o tumors extracted at indicated times in ( E ). ns = not significant; nd = not determined. G CSC frequencies determined using the extreme limiting dilution analysis algorithm ( http://bioinf.wehi.edu.au/software/elda/index.html ) (left, 95% CI) and images of resected tumors for the dilution 5 × 10 5 (right)

Article Snippet: For the detection of CD24, CD133 or CXCR4 cell surface marker expression, cells were incubated with a 1:5 dilution of a mouse anti-human CD24 PE (BD Cat no. 555428), a 1:50 dilution of a mouse anti-human CD133/1 Vio Bright R667 Ab (Miltenyi Cat no. 130–111–756) or a 1:50 dilution of a mouse anti-human CXCR4 (CD184) PE Ab (Miltenyi Cat no. 130–117–354).

Techniques: Control, Transfection, Flow Cytometry, Injection, Software

Journal: Bioactive Materials

Article Title: MSC-mimicking nanovesicle embedded bio-adhesive hydrogel for dual immunomodulation and osteogenesis to promote maxillofacial bone regeneration

doi: 10.1016/j.bioactmat.2026.02.032

Figure Lengend Snippet: nMSC@MT inheriting the original MSCs and could be effectively taken up by BMMSCs. a) Representative confocal images of PMVs with DiI-labeled cell membrane and Tht-labeled cytoplasm. Scale bar = 25 μm (above) and 1 μm (below). b) Illustration showing the co-extrusion procedure of nPMV and melatonin to obtain nMSC@MT. c) Representative TEM image of nMSC@MT. Scale bar = 100 nm. d, e) The sizes and the potential zetas of nPMV and nMSC@MT by DLS (n = 3). f, g) Coomassie brilliant blue and Western blot showing the preservation of stem cell markers, CD146, CD105, CD90, and functional protein, CXCR4, in PMV, nPMV, and nMSC@MT. h) DiI-labeled nMSC@MT (red) was detected in the cytoplasm of BMMSCs, suggesting the endocytosis of nMSC@MT. Scale bar = 10 μm. i) Immunofluorescence showing that a large amount of DiI-labeled nMSC@MT (red), released from the hydrogel, was taken up by CD146-labeled MSCs (green). Scale bar = 50 μm. j) BMMSCs uptake of DiI-labeled nMSC@MT in presence of blocking antibodies (anti-CXCR4) detected via flow cytometry. k) Analysis of the percentage of DiI positive cells in BMMSCs by flow cytometry (n = 3). l, m) Immunofluorescent staining images and corresponding semi-quantitative analysis (n = 3) of the uptake of DiI-labeled nPMV by BMMSCs in presence of blocking antibodies (anti-CXCR4). Scale bar = 50 μm. P-values are calculated using one-way ANOVA with Tukey's test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. not significant ( b was created with bioRender. com).

Article Snippet: To evaluate the involvement of CXCR4 in the uptake of nPMV, prior to co-culture with BMMSCs, nPMV was pre-treated with 160 nM CXCR4 blocking antibody (Ulocuplumab, Cat. HY-P99272, MedChemExpress, China) at room temperature for 1 h. Then nPMV was introduced into BMMSC cultures and incubated for 24 h. Subsequent analyses included flow cytometric analysis and immunofluorescence staining. nPMV without blocking antibody was served as a control.

Techniques: Labeling, Membrane, Western Blot, Preserving, Functional Assay, Immunofluorescence, Blocking Assay, Flow Cytometry, Staining

Journal: Bioactive Materials

Article Title: MSC-mimicking nanovesicle embedded bio-adhesive hydrogel for dual immunomodulation and osteogenesis to promote maxillofacial bone regeneration

doi: 10.1016/j.bioactmat.2026.02.032

Figure Lengend Snippet: nMSC@MT inheriting the original MSCs and could be effectively taken up by BMMSCs. a) Representative confocal images of PMVs with DiI-labeled cell membrane and Tht-labeled cytoplasm. Scale bar = 25 μm (above) and 1 μm (below). b) Illustration showing the co-extrusion procedure of nPMV and melatonin to obtain nMSC@MT. c) Representative TEM image of nMSC@MT. Scale bar = 100 nm. d, e) The sizes and the potential zetas of nPMV and nMSC@MT by DLS (n = 3). f, g) Coomassie brilliant blue and Western blot showing the preservation of stem cell markers, CD146, CD105, CD90, and functional protein, CXCR4, in PMV, nPMV, and nMSC@MT. h) DiI-labeled nMSC@MT (red) was detected in the cytoplasm of BMMSCs, suggesting the endocytosis of nMSC@MT. Scale bar = 10 μm. i) Immunofluorescence showing that a large amount of DiI-labeled nMSC@MT (red), released from the hydrogel, was taken up by CD146-labeled MSCs (green). Scale bar = 50 μm. j) BMMSCs uptake of DiI-labeled nMSC@MT in presence of blocking antibodies (anti-CXCR4) detected via flow cytometry. k) Analysis of the percentage of DiI positive cells in BMMSCs by flow cytometry (n = 3). l, m) Immunofluorescent staining images and corresponding semi-quantitative analysis (n = 3) of the uptake of DiI-labeled nPMV by BMMSCs in presence of blocking antibodies (anti-CXCR4). Scale bar = 50 μm. P-values are calculated using one-way ANOVA with Tukey's test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. not significant ( b was created with bioRender. com).

Article Snippet: To evaluate the involvement of CXCR4 in the uptake of nPMV, prior to co-culture with BMMSCs, nPMV was pre-treated with 160 nM CXCR4 blocking antibody (Ulocuplumab, Cat. HY-P99272, MedChemExpress, China) at room temperature for 1 h. Then nPMV was introduced into BMMSC cultures and incubated for 24 h. Subsequent analyses included flow cytometric analysis and immunofluorescence staining. nPMV without blocking antibody was served as a control.

Techniques: Labeling, Membrane, Western Blot, Preserving, Functional Assay, Immunofluorescence, Blocking Assay, Flow Cytometry, Staining

Journal: Research

Article Title: High Mobility Group Protein B1 Promotes Interferon Regulatory Factor 1 SUMOylation to Prime Trained Immunity of Circulating Monocytes and Aggravate the Progressive Synovial Inflammation in Knee Osteoarthritis

doi: 10.34133/research.1243

Figure Lengend Snippet: High mobility group protein B1 (HMGB1) promotes interferon regulatory factor 1 (IRF1) SUMOylation via MyD88 to induce trained immunity in knee osteoarthritis (KOA) monocytes. (A) Western blotting (WB) was used to detect the expression levels of IRF1 and MyD88 proteins in peripheral blood monocytes from patients in the healthy donor (HD) and KOA groups ( n = 3). (B) WB was used to detect the expression levels of IRF1 and MyD88 proteins in peripheral blood monocytes from mice in the negative control (NC) and HMGB1 groups ( n = 3). (C) Coimmunoprecipitation (CO-IP) was used to examine the interaction between IRF1 and MyD88 proteins in peripheral blood monocytes from patients in the HD and KOA groups. (D) CO-IP was used to examine the interaction between IRF1 and MyD88 proteins in peripheral blood monocytes from mice in the NC and HMGB1 groups. (E) A laser confocal microscope was used to observe the interaction between IRF1 and MyD88 proteins in peripheral blood monocytes from mice in the NC and HMGB1 groups. (F) CO-IP was used to evaluate the SUMOylation of IRF1 in peripheral blood monocytes from patients in the HD and KOA groups. (G) CO-IP was used to evaluate the SUMOylation of IRF1 in peripheral blood monocytes from mice in the NC and HMGB1 groups. (H) CO-IP was used to compare the differences in SUMOylation levels of IRF1 in peripheral blood monocytes from wild-type and MyD88 knockout mice after HMGB1 stimulation. (I) Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in peripheral blood monocytes from wild-type and MyD88 knockout mice after HMGB1 stimulation ( n = 6). (J) Flow cytometry was used to determine the proportion of CXCR4-positive cells in peripheral blood monocytes from wild-type mice stimulated with HMGB1 ( n = 3). (K) Flow cytometry was used to determine the proportion of CXCR4-positive cells in peripheral blood monocytes from MyD88 knockout mice stimulated with HMGB1 ( n = 3). Statistical results are represented by mean ± standard error of the mean (mean ± SEM), * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For the CXCR4 inhibitor intervention, mice were injected with plerixafor (HY-10046, MCE, USA) at a dose of 5 mg/kg into the tail vein.

Techniques: Western Blot, Expressing, Negative Control, Co-Immunoprecipitation Assay, Microscopy, Knock-Out, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Research

Article Title: High Mobility Group Protein B1 Promotes Interferon Regulatory Factor 1 SUMOylation to Prime Trained Immunity of Circulating Monocytes and Aggravate the Progressive Synovial Inflammation in Knee Osteoarthritis

doi: 10.34133/research.1243

Figure Lengend Snippet: Inhibition of high mobility group protein B1 (HMGB1) eliminated the trained immunity of circulating monocytes and their aggregation to knee osteoarthritis (KOA) synovial tissue. (A) In vivo imaging was used to observe the locations of cell migration in the KOA group, the KOA group with transfused fluorescently labeled trained circulating monocytes, and the KOA group with transfused fluorescently labeled untrained circulating monocytes in mice ( n = 3). (B) Enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in mouse peripheral serum at different time points under Nab-HMGB1 intervention ( n = 6). (C) Western blotting (WB) was used to detect the expression of IL-1β, IL-6, and TNF-α in mouse synovial tissue at different time points following Nab-HMGB1 intervention. (D) Representative WB bands showing the levels of IL-1β, IL-6, and TNF-α in peripheral circulating monocytes from mice at different time points under Nab-HMGB1 intervention ( n = 3). (E) Statistical graphs of the WB results for IL-1β, IL-6, and TNF-α levels in peripheral circulating monocytes from mice at different time points under Nab-HMGB1 intervention. (F) Representative WB bands showing the levels of IL-1β, IL-6, and TNF-α in peripheral circulating monocytes from mice at different time points under Nab-HMGB1 intervention ( n = 3). (G) Immunofluorescence was used to observe the numbers of CXCR4 + and Ly6c + cells in synovial tissue from the negative control (NC) group, KOA group, and Nab-HMGB1 group of mice. (H) The number of CXCR4 + circulating monocytes in mouse synovial tissue from the NC group, KOA group, and Nab-HMGB1 group was detected using magnetic bead sorting of mouse circulating monocytes followed by flow cytometry ( n = 3). Statistical results are represented by mean ± standard error of the mean (mean ± SEM), * P < 0.05, ** P < 0.01, *** P < 0.001. NS, not significant.

Article Snippet: For the CXCR4 inhibitor intervention, mice were injected with plerixafor (HY-10046, MCE, USA) at a dose of 5 mg/kg into the tail vein.

Techniques: Inhibition, In Vivo Imaging, Migration, Labeling, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Immunofluorescence, Negative Control, Flow Cytometry

Journal: Research

Article Title: High Mobility Group Protein B1 Promotes Interferon Regulatory Factor 1 SUMOylation to Prime Trained Immunity of Circulating Monocytes and Aggravate the Progressive Synovial Inflammation in Knee Osteoarthritis

doi: 10.34133/research.1243

Figure Lengend Snippet: CXCR4-dependent migration of trained subsets promoted synovial inflammation in knee osteoarthritis (KOA). (A) Flow cytometry was used to assess the effects of high mobility group protein B1 (HMGB1), CCL4, and interleukin-18 (IL-18) treatments on the proportion of CXCR4-positive cells among circulating monocytes in mice ( n = 3). (B) CXCR4 + cells were isolated using flow cytometric sorting. (C) Principal component analysis (PCA) was performed on CXCR4 + and CXCR4 − cells. (D) A volcano plot displays the number of differentially expressed genes between CXCR4 + and CXCR4 − cells. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) database enrichment analysis was conducted for the differentially expressed genes between CXCR4 + and CXCR4 − cells. (F) In vivo animal fluorescence imaging demonstrates the effects of transfusing CXCR4 + cells and administering the CXCR4 receptor antagonist plerixafor on CXCR4 + cells in KOA mice ( n = 3). (G) Hematoxylin and eosin (HE) staining was used to observe synovial pathology in the KOA group, the CXCR4 + cell transfusion group, and the CXCR4 + cell transfusion plus plerixafor group. (H) Representative Western blot images show protein expression levels of IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) in the synovial tissue of mice from the KOA group, CXCR4 + cell transfusion group, and CXCR4 + cell transfusion plus plerixafor group. (I) Quantitative Western blot analysis of IL-1β, IL-6, and TNF-α protein expression levels in synovial tissue from the 3 groups ( n = 3). (J) Quantitative polymerase chain reaction (PCR) analysis of IL-1β, IL-6, and TNF-α mRNA expression levels in synovial tissue from the KOA group, CXCR4 + cell transfusion group, and CXCR4 + cell transfusion plus plerixafor group ( n = 3). Statistical results are represented by mean ± standard error of the mean (mean ± SEM), ** P < 0.01, *** P < 0.001.

Article Snippet: For the CXCR4 inhibitor intervention, mice were injected with plerixafor (HY-10046, MCE, USA) at a dose of 5 mg/kg into the tail vein.

Techniques: Migration, Flow Cytometry, Isolation, In Vivo, Fluorescence, Imaging, Staining, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: Advanced Science

Article Title: Integrated Single‐Cell and Spatial Analysis Reveals a Metabolic‐Immune Axis Driving Aortic Dissection

doi: 10.1002/advs.75509

Figure Lengend Snippet: Lentivirus‐mediated Eno1 knockdown attenuates disease progression in AAD mouse models. (A) Images showing the characteristics of aortas from four different mouse model groups: Normal (control group), AAD (BAPN+Ang‐II‐induced AAD model), AAD‐lv‐sh‐NC (empty vector transfection+Ang‐II‐induced AAD model), and AAD‐lv‐sh‐ Eno1 ( Eno1 knockdown mouse model of AAD induced by BAPN and Ang‐II). (B) Maximal abdominal aortic outer diameters in the four groups. (C) Incidence of AAD in the four groups. n = 10 per group. (D) Survival curves for mice in the four different mouse model groups. (E) H&E staining of aortic tissues from the four different model groups. (F,G) Western blot analysis of key proteins involved in contractile SMC (α‐SMA), synthetic SMC (Opn, Mmp9), and SMC_C1_CP+ (Ceruloplasmin, Vegfa) in aortic tissues from the four different model groups. (H) Immunohistochemistry showing synthetic smooth muscle marker Opn and SMC_C1_CP+ markers (Ceruloplasmin, Vegfa) in aortic tissues from the four different model groups. (I) Western blot analysis of Mif and its receptors in aortic tissues from the four different model groups. (J) Immunofluorescence showing Mif and Cxcr4 expression in aortic tissues from the four different model groups (Mif: green, Cxcr4: red). Scale bar = 50 µm. (K) Expression of anti‐inflammatory ( iNos , Cd38 ) and pro‐inflammatory factors ( Cd206 ) in aortic tissues from the four different model groups. (L) Expression of pro‐inflammatory ( Tnf‐α , Il‐6 , Il‐1β ) and anti‐inflammatory markers ( Il‐10 ) from the four different model groups. (M) Flow cytometry analysis of the ratio of Cd206+Cd86‐ M2 macrophages and Cd86+Cd206− M1 macrophages in aortic tissues from the four different model groups. For panels B, G, I, K, L and M, ANOVA followed by Tukey's post hoc test was performed for comparisons among multiple groups. *** p < 0.001; ## p < 0.01.

Article Snippet: In some experiments, THP‐1 macrophages were treated with 2 μg/mL CXCR4 antagonist AMD3100 (HY‐10046, MedChem Express, Monmouth Junction, USA), 10 ng/ml recombinant human MIF protein (r MIF ; 10246‐H08H, Sino Biological, Beijing, China), or vehicles for 24 h before functional evaluations.

Techniques: Knockdown, Biomarker Discovery, Control, Plasmid Preparation, Transfection, Staining, Western Blot, Immunohistochemistry, Marker, Immunofluorescence, Expressing, Flow Cytometry