Journal: Materials today. Bio

Article Title: Precise delivery of doxorubicin and imiquimod through pH-responsive tumor microenvironment-active targeting micelles for chemo- and immunotherapy.

doi: 10.1016/j.mtbio.2022.100482

Figure Lengend Snippet: Fig. 10. Immunostaining of tumor tissues in tumor-bearing mice after treatments. (A) Immunohistochemistry images of tumor sections stained with CD3, CD8, and TNF-α antibodies. The scale bar is 100 μm. (B) Immunofluorescence images of tumor tissues after treatments for 12 days. The scale bar is 50 μm. Blue fluorescence represents the cell nucleus stained with DAPI. Green fluorescence represents the iNOS stained with the iNOS antibody conjugated with FITC.

Article Snippet: After 30 min, the tissue slice was stained with diluted iNOS antibody (Miltenyi Biotec, catalog: 130-116-357) at 4 C overnight.

Techniques: Immunostaining, Immunohistochemistry, Staining

Journal: Journal of Nanobiotechnology

Article Title: Macrophage biomimetic nanoparticle-targeted functional extracellular vesicle micro-RNAs revealed via multiomics analysis alleviate sepsis-induced acute lung injury

doi: 10.1186/s12951-024-02597-z

Figure Lengend Snippet: Inhibition of TLR, CXCR2, or Tnfα reduces the oxidative stress in the lung caused by EVs derived from LPS-stimulated macrophages. ( A ) The network of upregulated genes in neutrophils treated with LPS-BMDM-EVs relative to the control group. ( B ) The mRNA levels (mean ± SD, n = 3 per group) of TNF, CXCL10, Ifit1, IRF7, and Oasl1 in BMDNs from the control and LPS-BMDM-EV groups. *** P < 0.001 versus control. ( C ) The expression levels of NF-κB, p-NF-κB, Tnfα, CXCR2, NOX4, and iNOS (mean ± SD, n = 3 per group) of BMDNs stimulated with LPS-BMDM-EVs and then treated with CL-380,803, SB225002, or leonurine hydrochloride (LH). * P < 0.05 versus control; & P < 0.05 versus LPS-BMDM-EVs; # P < 0.05 versus LPS-BMDM-EVs; and $ P < 0.05 versus LPS-BMDM-EVs. ( D ) H&E staining and expression levels of MPO, NOX4, Tnfα, and iNOS in the lungs of the BMDM-EV treatment group after administration of CL-380,803, SB225002, or LH by tail vein injection ( n = 6 per group); scale bar: 100 μm. BMDM: Bone marrow-derived macrophages, BMDN: Bone marrow-derived neutrophils, and H&E: hematoxylin and eosin

Article Snippet: Clodronate liposomes were obtained from Vrije Universiteit Amsterdam (40337ES10; Netherlands); rabbit polyclonal CD63 (ab10895), CD9 (ab92726), Alix (ab117600), Tsg101 (ab30871), Ly6G (ab25377), and CXCR2 (ab14953) were sourced from Abcam (Cambridge, UK); and specific antibodies against CD81 (18250-1-AP), CD68 (28058-1-AP), Tnfaip3 (15104-1-AP), GAPDH (10494-1-AP), NF-κB (10745-1-AP), MPO (22225-1-AP), NOX4 (14374-1-AP), iNOS (18985-1-AP), and Tnfα (17590-1-AP) were obtained from Proteintech (Rosemont, IL, USA).

Techniques: Inhibition, Derivative Assay, Control, Expressing, Staining, Injection

Journal: Human Cell

Article Title: Single-cell dynamic RNA and glycosylation sequencing reveals the mechanism underlying the differentiation of pluripotent stem cells into hematopoietic stem cells

doi: 10.1007/s13577-025-01234-7

Figure Lengend Snippet: The analysis for the heterogeneity of endothelial clusters. a . The gene expression of endothelial, angiogenesis, and hematopoiesis in new RNA. b . The hotspot analyzed the similarity of cells and obtained 12 modules. c–d . The vascular endothelial cell migration pathway was selected from modules 3 and 8 and mapped to UMAP plots. e . The GO analysis of differential genes in high-glucose and low-glucose cells. f . The endothelial and hematopoietic genes in high-glucose and low-glucose cells in clusters 1 and 15

Article Snippet: From day 2 to day 6, 50 ng/mL human vascular endothelial growth factor (VEGF, MCE Catalog #HY- P70458 ), 50 ng/mL human basic fibroblast growth factor (bFGF, MCE Catalog #HY-P7331), 5 ng/mL BMP4, and 10 μM SB-431542 (MCE Catalog #HY-10431) were added to the BDM.

Techniques: Gene Expression, Migration

Journal: Human Cell

Article Title: Single-cell dynamic RNA and glycosylation sequencing reveals the mechanism underlying the differentiation of pluripotent stem cells into hematopoietic stem cells

doi: 10.1007/s13577-025-01234-7

Figure Lengend Snippet: Potential linkages of endothelial subsets in their differentiation fate. a . Clusters 1 and 15 were reclustered to seven new clusters, which were identified by differentially expressed genes, including NEFH, CLDN5, TOP2 A, RUNX1, MCM3, ANGPT2, and IGFBP3. b . The expression of endothelial and hematopoietic-related genes in endothelial subsets. c–d . The NTR and the levels of glycation in endothelial subsets. e . The expression of transcription factors in the endothelial subsets. f . The correlation between clusters 1, 15, and the endothelial subsets using the Spearman correlation analysis

Article Snippet: From day 2 to day 6, 50 ng/mL human vascular endothelial growth factor (VEGF, MCE Catalog #HY- P70458 ), 50 ng/mL human basic fibroblast growth factor (bFGF, MCE Catalog #HY-P7331), 5 ng/mL BMP4, and 10 μM SB-431542 (MCE Catalog #HY-10431) were added to the BDM.

Techniques: Expressing

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: NOS2-expressing macrophages is associated with response to αOX40 therapy (A) UMAP of monocytes/macrophages subclusters from scRNA-seq data in αOX40-treated MC38-bearing mice. (B) Representative marker genes in the monocyte/macrophage subclusters. (C) Pie chart showing the proportional distribution of monocyte/macrophage subsets of responders and nonresponders. (D) QuSAGE pathway analysis demonstrated enrichment of innate immune and phagocytic signaling pathways in distinct monocyte/macrophage subsets. (E) UMAP showing Mac_C1 signature genes and a heatmap of immune-related gene expression across TAM subclusters ( Z score normalized). (F) Violin plots comparing Nos2 expression levels in Mac_C1 subset between responsive and nonresponsive. (G) Flow cytometry analysis shows the percentage of M1-like (F4/80 + NOS2 + ) and M2-like (F4/80 + CD206 + ) macrophages in tumor tissues of control ( n = 5 mice), nonresponders (with minimal to no response, n = 4 mice), and responders (with a robust therapeutic response, n = 4 mice). (H and I) Comparison of Nos2 expression levels in responders versus nonresponders pre- or post-αOX40 treatment. Bilateral-MC38-bearing mice were treated with αOX40, and tumors from one side were analyzed by RNA-seq prior to (H) or following αOX40 treatment (I). The Nos2 expression was analyzed from RNA-seq data (left) and validated by RT-qPCR (right) ( n = 5 biological replicates). (J) NOS2 expression in tumor biopsies post-treatment determined by RNA-seq. Patients with advanced solid tumors and >1 prior therapy received HFB301001 monotherapy. Tumor biopsy samples were obtained on day 8 of cycle 2 for subsequent RNA-seq analysis. NOS2 expression were compared between patients achieving stable disease (SD, n = 3) and those with progressive disease (PD, n = 3). (K) GO enrichment analysis of upregulated genes in Mac_C1 of responders. (L) Calreticulin expression was quantified by flow cytometry in different response groups following αOX40 treatment ( n = 3 mice per group). (M) NOS2 expression in BMDMs was analyzed by flow cytometry after stimulation with CD8 + T cell supernatant and MC38 lysate, combined with TLR inhibition and IFN-γ blockade ( n = 5 biological replicates). (N) Quantification of Nos2 expression in BMDM by RT-qPCR after 24-h stimulation with MPLA (TLR4 agonist, 100 ng/mL), IFN-γ (20 ng/mL), or both. Data normalized to Gapdh and presented as fold-change relative to unstimulated controls ( n = 4 biological replicates). Data are shown as means ± SD from one of two independent experiments (G, H, I, L, M, and N). Statistical significance was determined using one-way ANOVA with multiple comparisons (G, L, M, and N) or using an unpaired two-tailed t test (H, I, and J). n.s., not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; VST, variance stabilized transformation; sup., supernatant; lys., tumor lysate; inh., inhibitor.

Article Snippet: To investigate whether macrophages tumoricidal effect is dependent on NOS2, 200 μM NOS2 inhibitor L-NIL (HY-12116, MCE) was added to the medium of cocultured cells.

Techniques: Expressing, Marker, Protein-Protein interactions, Gene Expression, Flow Cytometry, Control, Clinical Proteomics, Comparison, RNA Sequencing, Quantitative RT-PCR, Inhibition, Two Tailed Test, Transformation Assay

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: NOS2-high macrophages are significantly associated with Combo treatment efficacy (A) UMAP of macrophage subclusters from scRNA-seq data of MC38-bearing mice treated with control, MPLA+IFN-γ, αOX40, or Combo. Cells are color-coded by annotated subtype. (B) Bubble chart showing the top variable marker genes for identified macrophage subclusters. (C) Pie chart shows the relative abundance of four macrophage subclusters in control, αOX40, MPLA+IFN-γ, or Combo. (D) GO pathway analysis identifying significantly enriched signaling pathways in the Mac_S2 subcluster compared to other macrophage subpopulations. (E) Violin plots showing Nos2 expression levels across macrophage subclusters. (F) Violin plots comparing Nos2 and Cd206 expression levels among different treatment groups. (G) Frequency of M1-like, M2-like, or the ratio of M1/M2-like macrophage cells in tumor tissues from control, αOX40, MPLA+IFN-γ, and Combo groups with two time points, as determined by flow cytometry ( n = 5–10 mice per group). (H) Multiple immunofluorescence signal intensities of NOS2 + F4/80 + and CD206 + F4/80 + cells in the TME of control, αOX40, MPLA+IFN-γ, and Combo groups. Scale bars, 20 μm. Data are shown as means ± SD from one of two independent experiments (G and H). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test (G). n.s., not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: To investigate whether macrophages tumoricidal effect is dependent on NOS2, 200 μM NOS2 inhibitor L-NIL (HY-12116, MCE) was added to the medium of cocultured cells.

Techniques: Control, Marker, Protein-Protein interactions, Expressing, Flow Cytometry, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: NOS2-dependent direct tumor cell killing by macrophages in Combo therapy (A) Schematic of co-culture using CFSE-labeled MC38 cells with tumor- or spleen-derived macrophages, with/without NIL treatment. (B) Flow cytometry quantification of 7-AAD + MC38 cells after 48 h co-culture with tumor-(right) or spleen (left)-derived macrophages ( n = 3 biological replicates). (C) Quantification of 7-AAD + MC38 cells after 48 h in vitro co-culture with or without NIL treatment ( n = 3 biological replicates). (D) Treatment schedule for MC38- or B16-tumor-bearing Nos2 KOor WT mice treated with Combo ( n = 6 mice per group). (E and F) Survival curves of MC38-bearing (E) and B16-bearing mice (F) were analyzed using the log rank test. (G) Flow cytometry analysis of 7-AAD + MC38 cells after 48-h co-culture with CFSE-labeled MC38 cells and BMDMs from Nos2 KO mice ( n = 5 biological replicates). (H) Phagocytosis rate of MC38 cells engulfed by BMDMs was assessed by flow cytometry ( n = 3 biological replicates). (I) Surface expression of CRT on MC38 cells was assessed by flow cytometry after co-culture with MPLA- and IFN-γ-polarized BMDMs in vitro ( n = 3 biological replicates). (J) Analysis of CALR + MC38 cells from MC38-tumor-bearing mice following final treatment with control or Combo, assessed by flow cytometry ( n = 5 biological replicates). Data are shown as means ± SD from one of two independent experiments (B, C, E, F, G, H, I, and J). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons (B, C, and I) or using unpaired Student’s t test (G, H, and J). Log rank tests (E and F) were also used for statistical analysis. n.s., not significant; ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Combo, MPLA, and IFN-γ combined with αOX86 (E and F).

Article Snippet: To investigate whether macrophages tumoricidal effect is dependent on NOS2, 200 μM NOS2 inhibitor L-NIL (HY-12116, MCE) was added to the medium of cocultured cells.

Techniques: Co-Culture Assay, Labeling, Derivative Assay, Flow Cytometry, In Vitro, Expressing, Control

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: Foxp3 + Treg depletion and macrophage reprogramming are involved in the anti-tumor effect of Combo (A) Flow cytometry analysis of TME. MC38-tumor-bearing mice were treated with control, αOX40, MPLA+IFN-γ, or Combo for two and three doses, and tumors were analyzed by flow cytometry. (B) Frequency and absolute count of CD25 + FOXP3 + cells in tumor tissues from control, αOX40, MPLA+IFN-γ, and Combo groups with two time points, as determined by flow cytometry ( n = 5 mice per group). (C) Treatment schedule for MC38-tumor-bearing Fcer1g KO or FcγRIIb KO mice. Mice were treated with Control, αOX40, MPLA+IFN-γ, and Combo every 3 days for a total of four doses. (D and E) Survival curves of Fcgr1g KO (D) and FcgrIIb KO (E) mice following treatment ( n = 5–6 mice per group) were monitored. (F) Treatment schedule. MC38-tumor-bearing mice were treated with MPLA and IFN-γ in combination with either OX40-mIgG2a or OX40-hIgG1 agonist antibodies (top), and the corresponding survival curves are shown (bottom) ( n = 5–7 mice per group). (G) Schematic of the co-culture experiment involving BMDMs and Tregs at a ratio of 1:4 (BMDM:Treg); Nos2 expression was measured by RT-qPCR. (H) Relative expression of Nos2 following the co-culture ( n = 4 biological replicates). (I) Multiple immunofluorescence (mIF) staining of MC38 tumors from mice treated with control, αOX40, MPLA+IFN-γ, or Combo, showing FOXP3 and NOS2 expression in border or intra-tumoral. Scale bars, 50 μm. (J) Analysis of cell numbers of FOXP3 and NOS2 expression at the border and intra-tumoral. Representative images from five randomly chosen fields were quantified with ImageJ. Data are shown as means ± SD from one of two independent experiments (B, D, E, F, H, and I). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons (B and H). Log rank test was also used (D–F). n.s., not significant; ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Combo, MPLA, and IFN-γ combined with αOX86 (C–E).

Article Snippet: To investigate whether macrophages tumoricidal effect is dependent on NOS2, 200 μM NOS2 inhibitor L-NIL (HY-12116, MCE) was added to the medium of cocultured cells.

Techniques: Flow Cytometry, Control, Co-Culture Assay, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: International journal of molecular medicine

Article Title: Naringenin modulates the NO‑cGMP‑PKG signaling pathway by binding to AKT to enhance osteogenic differentiation in hPDLSCs.

doi: 10.3892/ijmm.2024.5391

Figure Lengend Snippet: Figure 3. Ability of NAR to promote osteogenesis in human periodontal ligament stem cells is mitigated after L‑NAME (eNOS inhibitor) treatment. (A) Alkaline phosphatase staining in different treatment groups. (B) Alizarin red staining in different treatment groups. (C) NO levels in different treatment groups. (D) Gene expression levels of RUNX2, OPN and sGC in different treatment groups. (E‑G) Protein expression of RUNX2, OPN, sGC and p‑eNOS in various treatment groups. *P<0.05, **P<0.01, ***P<0.001. NAR, naringenin; eNOS, endothelial nitric oxide synthase; RUNX2, Runt‑related transcription factor; OPN, osteopontin; sGC, soluble guanylate cyclase; NO, nitric oxide; p‑, phosphorylated; t‑, total; L‑NAME, NG‑nitro‑L‑arginine methyl ester.

Article Snippet: Cells treated with NAR (10 μmol/l) were also treated with NG‐nitro‐L‐arginine methyl ester (L‐NAME; 70 μM) to inhibit endothelial nitric oxide synthase (eNOS), 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 20 μM) to inhibit soluble guanylate cyclase (sGC) and AKT inhibitor VIII (58 nM) to inhibit AKT, as recommended by the manufacturer (MedChemExpress for all).

Techniques: Staining, Gene Expression, Expressing

Journal: International journal of molecular medicine

Article Title: Naringenin modulates the NO‑cGMP‑PKG signaling pathway by binding to AKT to enhance osteogenic differentiation in hPDLSCs.

doi: 10.3892/ijmm.2024.5391

Figure Lengend Snippet: Figure 6. Impact of AKT inhibitor VIII on NAR‑mediated osteogenic promotion in human periodontal ligament stem cells. (A) Alkaline phosphatase staining in different treatment groups. (B) Alizarin red staining in different treatment groups. (C) Gene expression of RUNX2 and OPN in different treatment groups. (D‑F) Protein levels of RUNX2, OPN, p‑AKT and p‑eNOS in different treatment groups. *P<0.05, **P<0.01, ***P<0.001. NAR, naringenin; RUNX2, Runt‑related transcription factor; OPN, osteopontin; eNOS, endothelial nitric oxide synthase; p‑, phosphorylated; t‑, total.

Article Snippet: Cells treated with NAR (10 μmol/l) were also treated with NG‐nitro‐L‐arginine methyl ester (L‐NAME; 70 μM) to inhibit endothelial nitric oxide synthase (eNOS), 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 20 μM) to inhibit soluble guanylate cyclase (sGC) and AKT inhibitor VIII (58 nM) to inhibit AKT, as recommended by the manufacturer (MedChemExpress for all).

Techniques: Staining, Gene Expression

Journal: International journal of molecular medicine

Article Title: Naringenin modulates the NO‑cGMP‑PKG signaling pathway by binding to AKT to enhance osteogenic differentiation in hPDLSCs.

doi: 10.3892/ijmm.2024.5391

Figure Lengend Snippet: Figure 8. Schematic diagram of the mechanism by which naringenin promotes the osteogenic differentiation of human periodontal ligament stem cells. eNOS, endothelial nitric oxide synthase; p, phosphorylated; NO, nitric oxide; sGC, soluble guanylate cyclase; cGMP, cyclic guanosine monophosphate; TRPC6, transient receptor potential cation channel, subfamily C, member 6; PKG, protein kinase G.

Article Snippet: Cells treated with NAR (10 μmol/l) were also treated with NG‐nitro‐L‐arginine methyl ester (L‐NAME; 70 μM) to inhibit endothelial nitric oxide synthase (eNOS), 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 20 μM) to inhibit soluble guanylate cyclase (sGC) and AKT inhibitor VIII (58 nM) to inhibit AKT, as recommended by the manufacturer (MedChemExpress for all).

Techniques: