Journal: Molecular Therapy. Nucleic Acids

Article Title: Nitric oxide-dependent stabilization of vimentin confers chemoresistance in ovarian cancer

doi: 10.1016/j.omtn.2026.102924

Figure Lengend Snippet: Constitutive iNOS expression in ovarian cancer cells (A) mRNA expression levels of iNOS across an ovarian cancer cell lines from the CCLE database. (B) Assessment of the relative NOS2 mRNA expression in ovarian cancer cell lines from the NCI-60 database. (C) Protein expression levels of iNOS in ovarian cancer cells were determined by western blot. (D) RT qPCR analyses of NOS2 mRNA in ovarian cancer cell lines. (E) Constitutive protein expression of iNOS is maintained regardless of stimulation with LPS or a cytokine mix in OVCAR8 and A2780 cells. (F) Inducible expression of iNOS upon cytokine stimulation in A2780 wild-type cells lacks basal expression. Experiments were performed in biological duplicate or triplicate.

Article Snippet: The following primers for TaqMan Gene Expression Assay were purchased from Thermo Fisher Scientific; human VIM (Cat. #Hs00958111_m1) and human NOS2 (Cat. #Hs01075529_m1).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: Molecular Therapy. Nucleic Acids

Article Title: Nitric oxide-dependent stabilization of vimentin confers chemoresistance in ovarian cancer

doi: 10.1016/j.omtn.2026.102924

Figure Lengend Snippet: iNOS increases sensitivity to cisplatin in ovarian cancer (A) The cell viability of OVCAR8 cells was assessed using RealTime-Glo MT Cell Viability Assay after culturing in increasing concentrations of cisplatin for 72 h. (B) OVCAR8 cells incubated with or without L-NMMA for 48 h, measured by RealTime-Glo MT Cell Viability Assay. (C) Cell viability after either no treatment (control), cisplatin, and a combination of cisplatin and L-NMMA by RealTime-Glo MT Cell Viability Assay. (D) Western blot analysis of vimentin protein expression in ovarian cancer cell lines. (E) Ovarian cancer cell lines were analyzed for VIM mRNA levels by qPCR. (F) OVCAR8 cells were treated with or without L-NMMA (4, 6, 8, and 10 mM) for 48 h, and western blot was used to analyze the effect of L-NMMA on the vimentin protein expression. B-actin was used as a loading control. Band densities were quantified using ImageJ analysis. Error bars, SEM. ∗ p < 0.05; ∗∗ p < 0.01 (compared with the control group, using two-way ANOVA). All experiments were independently repeated three times.

Article Snippet: The following primers for TaqMan Gene Expression Assay were purchased from Thermo Fisher Scientific; human VIM (Cat. #Hs00958111_m1) and human NOS2 (Cat. #Hs01075529_m1).

Techniques: Viability Assay, Incubation, Control, Western Blot, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Nitric oxide-dependent stabilization of vimentin confers chemoresistance in ovarian cancer

doi: 10.1016/j.omtn.2026.102924

Figure Lengend Snippet: iNOS knockout increased chemosensitivity and impaired cell motility and migration of ovarian cancer cells (A) Immunoblotting showing reduced iNOS (left) and vimentin (right) expression levels following NOS2 knockdown in the OVCAR8 cells. (B) NOS2 knockdown sensitized OVCAR8 cells to cisplatin, reducing its IC 50 value. Log-logistic model was used to analyze the data. The group comparison between control group and KO-1 had a p value = 8.98e-07. The comparison between control and KO-2 had a p value = 0.0132. The detected EC50 for control group was 0.8554, for KO-1 was 0.6037, for KO2 was 0.7688. (C and D) Analysis of reduced protein (left) and mRNA (right) expression of iNOS and vimentin following siRNA transfection in OVCAR8 and A2780cis cells for 72 h, assessed by western blot and RT-qPCR. (E) OVCAR8 cells were transfected with 2 different siRNAs or the scramble siRNA and were assessed for migration using the scratch wound assay. The area of the wound was measured at 0, 12, 24, and 36 h by the IncuCyte live-cell analysis system. Two-way repeated measures ANOVA was used to analyze the data. After 6 h, siRNA1 had p value = 1, siRNA2 had p value = 0.23. After 12 h, siRNA1 had p value = 0.71 and siRNA 2 had p value = 0.16. After 24 h, siRNA1 had a p value = 0.39 and siRNA2 had a p value = 0.05. After 36 h, siRNA1 had a p value = 0.86 and siRNA2 had a p value = 0.04. (F) NOS2 KO-1 and KO-2 OVCAR8 cells formed significantly fewer colonies compared to parental OVCAR8. The experiment was performed in triplicate with three biological replicates. Statistical analysis was conducted with two-way ANOVA. ∗ p < 0.05 and ∗∗ p < 0.01. (G) Silencing of NOS2 by two different siRNAs significantly reduced the number of colonies formed by OVCAR8 cells. Clonogenic growth was measured after 10 days, quantified using ImageJ, and represented as a bar graph (mean ± SEM). The experiment was performed in triplicate with three biological replicates. Statistical analysis was conducted with two-way ANOVA. ∗ p < 0.05 and ∗∗ p < 0.01. All experiments were independently repeated two to three times.

Article Snippet: The following primers for TaqMan Gene Expression Assay were purchased from Thermo Fisher Scientific; human VIM (Cat. #Hs00958111_m1) and human NOS2 (Cat. #Hs01075529_m1).

Techniques: Knock-Out, Migration, Western Blot, Expressing, Knockdown, Comparison, Control, Transfection, Quantitative RT-PCR, Scratch Wound Assay Assay, Cell Analysis

Journal: Molecular Therapy. Nucleic Acids

Article Title: Nitric oxide-dependent stabilization of vimentin confers chemoresistance in ovarian cancer

doi: 10.1016/j.omtn.2026.102924

Figure Lengend Snippet: Knockdown of NOS2 impaired the expression of genes involved in epithelial-to-mesenchymal transition (A) Heatmap showing genes whose expression differed significantly between NOS2 KO-1 and parental OVCAR8 cells. Top 15 altered genes are presented; red indicates upregulated genes, while green indicates downregulated genes. (B) Volcano plot showing the genes that altered significantly between NOS2 knockdown OVCAR8 and parental cells based on RNA-seq analysis. (C) Bubble plot of KEGG enrichment terms based on RNA-seq results showing enrichment of pathways related to wound healing, extracellular matrix organization, and regulation of cell shape. All the experiments were performed in triplicates.

Article Snippet: The following primers for TaqMan Gene Expression Assay were purchased from Thermo Fisher Scientific; human VIM (Cat. #Hs00958111_m1) and human NOS2 (Cat. #Hs01075529_m1).

Techniques: Knockdown, Expressing, RNA Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Nitric oxide-dependent stabilization of vimentin confers chemoresistance in ovarian cancer

doi: 10.1016/j.omtn.2026.102924

Figure Lengend Snippet: L-NMMA promotes vimentin destabilization by enhancing its ubiquitination (A) CHX chase assay showing vimentin protein levels in OVCAR8 cells treated with 10 mM L-NMMA at different time points. (B) Vimentin ubiquitination was assessed in OVCAR8 cells treated with L-NMMA for 24 h, in the presence of the proteasome inhibitor MG-132 (10 μM) for the final 4 h, followed by immunoprecipitation and western blot using an anti-ubiquitin antibody. (C) Measurement of vimentin S-nitrosylation levels was performed by immunoprecipitation in OVCAR8 cells. (D) Western blot analysis of vimentin expression in OVCAR8 cells treated with 10 mM L-NMMA alone or in combination with MG132 at 1, 5, or 10 μM. (E) Tumor growth and proliferation were monitored in mice bearing parental and NOS2 KO OVCAR8 tumors, evaluated by ROI measurements every 4 days ( n = 6). (F) Kaplan-Meier survival curves of mice bearing parental and NOS2 KO OVCAR8 tumors following cisplatin treatment ( n = 6). Data are presented as mean ± SEM. Statistical analysis was performed using two-way ANOVA for growth curves and the Kaplan-Meier method for survival analysis ( p < 0.05, ∗ p < 0.01). Experiments were performed in duplicate or triplicate.

Article Snippet: The following primers for TaqMan Gene Expression Assay were purchased from Thermo Fisher Scientific; human VIM (Cat. #Hs00958111_m1) and human NOS2 (Cat. #Hs01075529_m1).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, 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: Bioactive Materials

Article Title: Immunomodulatory effects of biodegradable Mg–Cu–Zn alloy in esophageal cancer

doi: 10.1016/j.bioactmat.2026.02.046

Figure Lengend Snippet: Distribution of CD163 + M2 TAMs in AKR-derived allograft tumor tissues from immunocompetent C57BL/6 mice. (a, b) Representative IHC staining images showing CD163 + M2 TAMs in the (a) peritumoral stroma and (b) tumor islets. Lower panels display higher-magnification views of the regions outlined by red dashed boxes. (c, d) Quantification of CD163 + cells in the (c) peritumoral stroma and (d) tumor islets. (e) Comparison of CD163 + cell density between the peritumoral stroma and tumor islets. (f) Total number of CD163 + cells in allograft tumors (peritumoral stroma and tumor islets combined). (g, h) Comparison of the density between iNOS + cells and CD163 + cells in the (g) peritumoral stroma and (h) tumor islets. (i, j) Quantification of iNOS + /CD163 + ratio in the (i) peritumoral stroma and (j) tumor islets. p < 0.05 (∗), p < 0.01 (∗∗), p < 0.001 (∗∗∗). A field of view is ∼0.086 mm 2 in (c−j).

Article Snippet: Tissue sections were then incubated with primary antibodies against iNOS (22226-1-AP, ProteinTech, China), CD163 (A26411PM, Abclone, China), CD8 (SP16, Maixin, China), CD4 (SP35, Maixin, China) or Ki-67 (12202S, Cell Signaling Technology) for 12 h at 4 °C, followed by secondary antibodies (Beyotime Biotechnology, Nantong, China).

Techniques: Derivative Assay, Immunohistochemistry, Comparison

Journal: Bioactive Materials

Article Title: Immunomodulatory effects of biodegradable Mg–Cu–Zn alloy in esophageal cancer

doi: 10.1016/j.bioactmat.2026.02.046

Figure Lengend Snippet: Distribution of CD163 + M2 TAMs in AKR-derived allograft tumor tissues from immunocompetent C57BL/6 mice. (a, b) Representative IHC staining images showing CD163 + M2 TAMs in the (a) peritumoral stroma and (b) tumor islets. Lower panels display higher-magnification views of the regions outlined by red dashed boxes. (c, d) Quantification of CD163 + cells in the (c) peritumoral stroma and (d) tumor islets. (e) Comparison of CD163 + cell density between the peritumoral stroma and tumor islets. (f) Total number of CD163 + cells in allograft tumors (peritumoral stroma and tumor islets combined). (g, h) Comparison of the density between iNOS + cells and CD163 + cells in the (g) peritumoral stroma and (h) tumor islets. (i, j) Quantification of iNOS + /CD163 + ratio in the (i) peritumoral stroma and (j) tumor islets. p < 0.05 (∗), p < 0.01 (∗∗), p < 0.001 (∗∗∗). A field of view is ∼0.086 mm 2 in (c−j).

Article Snippet: Tissue sections were then incubated with primary antibodies against iNOS (22226-1-AP, ProteinTech, China), CD163 (A26411PM, Abclone, China), CD8 (SP16, Maixin, China), CD4 (SP35, Maixin, China) or Ki-67 (12202S, Cell Signaling Technology) for 12 h at 4 °C, followed by secondary antibodies (Beyotime Biotechnology, Nantong, China).

Techniques: Derivative Assay, Immunohistochemistry, Comparison

Journal: Redox Biology

Article Title: Multi-omic analysis reveals nitric oxide dependent remodeling in classically activated macrophages and identifies negative regulation mediated by AKR1A1

doi: 10.1016/j.redox.2026.104181

Figure Lengend Snippet: Global proteomic profiling reveals NO•-dependent remodeling in classically activated macrophages (A) Nitrite concentrations in media of wild-type (WT) or iNOS knockout (KO) BMDM with or without LPS/IFNγ stimulation for 48 h, and with or without 200 μM DETA-NONOate treatment. Data represents mean ± standard deviation, n = 3 biological replicates. Statistical analysis by one-way ANOVA with Tukey's post hoc test for multiple comparisons with p-value reported; ns indicates not significant. (B) Principal component analysis (PCA) of all proteins. PC1 and PC2 shown with 95% confidence ellipses. (C – D) Pathway enrichment analysis (Mouse WikiPathway 2024 via Enrichr) for PC2-negative (C) and PC2-positive (D) contributors. (E) Heatmap showing Z-score normalized expression of all proteins across condition (individual replicates designated by "_1″ through "_4″ suffix). Hierarchical clustering based on Pearson correlation distance (Ward.D2 linkage). Color scale: blue (low) and red (high) expression relative to mean. Rows categorized by response pattern: NO-dependent upregulation (red; WT stimulation effect >0.4 AND NO-specific effect >0.5); NO-dependent downregulation (blue; WT stimulation effect < −0.4 AND NO-specific effect < −0.5); NO-independent response (yellow; |WT stimulation effect| > 0.4 AND |iNOS KO stimulation effect| > 0.4 AND |NO-specific effect| < 0.5); under thresholds (grey; not meeting criteria above).

Article Snippet: Primary bone marrow-derived macrophages (BMDMs) were isolated from 8 to 12-week-old male and female wild-type C57BL/6J mice or B6.129P2- Nos2 tm1Lau /J (iNOS KO) mice (Jackson Laboratory).

Techniques: Knock-Out, Standard Deviation, Expressing

Journal: Redox Biology

Article Title: Multi-omic analysis reveals nitric oxide dependent remodeling in classically activated macrophages and identifies negative regulation mediated by AKR1A1

doi: 10.1016/j.redox.2026.104181

Figure Lengend Snippet: The effect of NO• in transcriptomic remodeling and its role in mediating major proteomic changes. (A) Left : Cross-omic correlation analysis comparing iNOS-dependent proteomic and transcriptomic changes (log 2 fold change: stimulated iNOS KO versus stimulated WT) in RAW264.7 cells for genes with corresponding protein measurements (n = 5001). Points colored by concordance category based on statistical significance: protein only (blue), RNA only (orange), both significant with opposite direction (yellow), both significant with concordant effect (green), or neither (grey). Linear regression with 95% confidence interval (shaded region). Doughnut chart shows category distribution (%) with counts in each category. Right : Pathway enrichment analysis (Mouse WikiPathway 2024 via Enrichr) for each of the category. (B–C) Cross-omic correlation for PCA-defined NO-regulated protein subsets. (B) PC2-negative contributors. Points colored by pathway membership based on enrichment analysis: ETC/OXPHOS (red), Cell Cycle/IL-17A signaling (blue), or other pathways (grey). All pathway-annotated genes are labeled. (C) PC2-positive contributors. Points colored by pathway membership: Oxidative Stress/Glutathione metabolism (orange) or other pathways (grey). All pathway-annotated genes are labeled. Linear regression with 95% confidence interval shown (shaded region) for B–C. (D – E) Transcription factor pathway enrichment analysis (D: ChEA 2022; E: ENCODE and ChEA Consensus) using Enrichr among PC2-positive contributors from C.

Article Snippet: Primary bone marrow-derived macrophages (BMDMs) were isolated from 8 to 12-week-old male and female wild-type C57BL/6J mice or B6.129P2- Nos2 tm1Lau /J (iNOS KO) mice (Jackson Laboratory).

Techniques: Labeling

Journal: Redox Biology

Article Title: Multi-omic analysis reveals nitric oxide dependent remodeling in classically activated macrophages and identifies negative regulation mediated by AKR1A1

doi: 10.1016/j.redox.2026.104181

Figure Lengend Snippet: NO• drives complex-specific remodeling of the electron transport chain (A) Cross-model proteomic correlation for ETC/OXPHOS proteins comparing iNOS-dependent changes (log 2 fold change: stimulated iNOS knockout (KO) versus stimulated wildtype (WT)) between BMDM (x-axis) and RAW264.7 cells (y-axis). Dots represent 68 proteins defined by KEGG OXPHOS pathway that are quantified in both models, color coded by ETC Complex assignment; V-ATPase subunits (vacuolar/lysosomal) shown separately from mitochondrial Complex V (F-type ATP synthase). Linear regression with 95% confidence interval (shaded region). (B) Cross-omic correlation for ETC/OXPHOS proteins comparing iNOS-dependent transcriptomic (x-axis) and proteomic (y-axis) changes in RAW264.7 cells (n = 69 genes with measurements in both datasets). Points colored by ETC complex as in (A). Linear regression with 95% confidence interval shown. (C – G) Heatmaps showing changes in each of the ETC complexes: (C) Complex I, (D) Complex II, (E) Complex III, (F) Complex IV, (G) Complex V, across eight experimental conditions: unstimulated (unstim) or stimulated (stim) with LPS/IFNγ for 48 h (stim) in WT or iNOS KO genotypes; ± DETA-NONOate (DETA). Colors represent row-wise Z-score normalized protein abundance (blue = decreased, red = increased relative to row mean). Rows (proteins) clustered by Pearson correlation; columns ordered by experimental condition. n = 4 biological replicates per condition (individual replicates designated by “_1” through “_4” suffix). (H) . Normalized oxygen consumption rate (OCR) of BMDM across experimental conditions as described in C-G. Data represent mean ± standard deviation, n = 3 biological replicates. Statistical analysis by one-way ANOVA with Tukey's post hoc test for multiple comparisons with p-value reported; ns indicates not significant.

Article Snippet: Primary bone marrow-derived macrophages (BMDMs) were isolated from 8 to 12-week-old male and female wild-type C57BL/6J mice or B6.129P2- Nos2 tm1Lau /J (iNOS KO) mice (Jackson Laboratory).

Techniques: Knock-Out, Quantitative Proteomics, Standard Deviation

Journal: Redox Biology

Article Title: Multi-omic analysis reveals nitric oxide dependent remodeling in classically activated macrophages and identifies negative regulation mediated by AKR1A1

doi: 10.1016/j.redox.2026.104181

Figure Lengend Snippet: Akr1a1 induction requires both classical activation and NO• signaling and is partially NRF2-dependent (A) Proteins significantly decreased in iNOS KO vs WT (both LPS/IFNγ-stimulated) across all three datasets (BMDM proteomics, RAW264.7 proteomics, RAW264.7 RNA-seq), ranked by RAW264.7 protein fold change. Red highlights proteins significantly induced by LPS/IFNγ in WT cells (p-adj <0.05, log2FC > 0). Top 15 labeled. (B) Top : Representative immunoblot showing AKR1A1 and iNOS protein abundance in WT and iNOS KO BMDMs stimulated with LPS/IFNγ for 0, 6, 12, 24, or 48 h. Bottom : Quantification of AKR1A1 and iNOS protein abundance (normalized to α-tubulin). Data represents mean ± standard deviation (SD) from n = 3 independent experiments. (C) Relative abundance of AKR1A1 protein from RAW264.7 cell proteomics dataset. (D) Left: Immunoblot showing AKR1A1 and iNOS protein abundance in primary WT or iNOS KO peritoneal macrophages with or without 48-h LPS/IFNγ stimulation (in triplicate). Right: Quantification of AKR1A1 protein abundance (normalized to α-tubulin). Data represents mean ± SD, n = 3 biological replicates. (E) Left: Representative immunoblot showing AKR1A1 and iNOS protein abundance in WT and iNOS KO BMDMs under four conditions: treated or untreated for 48 h with LPS/IFNγ alone, 200 μM DETA-NONOate alone, or LPS/IFNγ and DETA-NONOate combined. Right: Quantification of AKR1A1 protein abundance (normalized to α-tubulin). Data represents mean ± SD, n = 3 independent experiments. (F) Relative mRNA expression of Akr1a1 measured by RT-PCR from cells treated as in E. Expression normalized to Hnrpab reference gene using ΔΔCt method. Data represents mean ± SD, n = 3 biological replicates. (G – I) Relative abundance of MafG (G), MafK (H), and MafF (I) from BMDM proteomics dataset. (J) Relative mRNA expression of Akr1a1 in iNOS KO BMDMs unstimulated or stimulated with LPS/IFNγ ± DETA-NONOate ± ML385 (Nrf2 inhibitor, 10 μM) for 48 h. Expression normalized to Hnrpab . Data represents mean ± SD, n = 3 biological replicates. Statistics : For panels B-J, statistical comparisons were performed using one-way ANOVA with Tukey's post hoc test for multiple comparisons with p-value reported; ns indicates not significant.

Article Snippet: Primary bone marrow-derived macrophages (BMDMs) were isolated from 8 to 12-week-old male and female wild-type C57BL/6J mice or B6.129P2- Nos2 tm1Lau /J (iNOS KO) mice (Jackson Laboratory).

Techniques: Activation Assay, RNA Sequencing, Labeling, Western Blot, Quantitative Proteomics, Standard Deviation, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Redox Biology

Article Title: Multi-omic analysis reveals nitric oxide dependent remodeling in classically activated macrophages and identifies negative regulation mediated by AKR1A1

doi: 10.1016/j.redox.2026.104181

Figure Lengend Snippet: Akr1a1 regulates metabolic and functional response to classical activation by counteracting NO• (A ) Schematic showing AKR1A1's main enzyme activity as SNO-CoA reductase (yellow) and its mechanistic connection to NO• driven inhibition of pyruvate dehydrogenase complex (PDHC) (blue). SNO-CoA is the key molecule delivering NO-derived modifications onto the lipoic cofactor at the catalytic center of PDHC's E2 subunits (B) Left: Representative immunoblot showing iNOS, DLAT (E2 subunit of PDHC), AKR1A1 protein abundance, along with functional lipoic acid detection (anti-lipoic acid antibody) at the molecular weight of DLAT in immortalized bone marrow-derived macrophages (iBMDMs). Wildtype (WT) or Akr1a1 KO (KO) iBMDMs were stimulated with LPS/IFNγ for 0, 24, or 48 h. Each lane represents an independent iBMDM clone (generated using independent guide RNAs for Akr1a1 KO). Right: Quantification of functional lipoic acid to DLAT protein ratio at 24-h timepoint, normalized to unstimulated WT condition. Data represents mean ± standard deviation (SD), n = 2 independent clones per genotype. (C) Relative PDHC enzymatic activity measured in cell lysates from iBMDMs treated as in (B) at 0-, 24-, and 48-h timepoints. Activity normalized to unstimulated WT. Data represents mean ± SD, n = 4 measurements per genotype per timepoint (2 independent iBMDM clones assayed in 2 separate experiments) . (D) Experimental design schematic for in vitro PDHC–Akr1a1 co-incubation activity assays. Purified porcine PDHC was incubated for 3 h at room temperature with indicated combinations of PDHC substrates (NADH and CoA) and PAPA-NONOate (NO• donor) ± purified Akr1a1-FLAG (WT or K127A) ± NADPH (Akr1a1 cofactor), followed by measurement of PDHC activity. (E) Relative PDHC enzymatic activity from in vitro co-incubation assays as described in (D). All components added simultaneously and incubated for 3 h at room temperature before saturating levels of substrate and activity measurement. Activity normalized to protein only control. Data represents mean ± SD, n = 3 independent reactions. (F) Schematic showing SNO-CoA mediated inhibition of PDHC upstream of IRG1 regulating itaconate level, and AKR1A1 counteracting the effect of NO in this process. (G) Percent 2-labeled acetyl-CoA from kinetic U– 13 C- d -glucose tracing in WT, Akr1a1 KO, or Akr1a1/iNOS DKO RAW264.7 cells unstimulated or stimulated with LPS/IFNγ for 48 h. Data represents mean ± SD, n = 3 biological replicates. (H) Relative total abundance of itaconate from conditions described in (G). (I) Pathway enrichment analysis (GO Biological Process) using Enrichr among downregulated differential expressed genes from . (J) Relative mRNA expression of IL6 from of WT, Akr1a1 KO, iNOS KO and Akr1a1/iNOS DKO RAW264.7 cells stimulated for 0-, 3-, 6-, 12, 24-, and 48-h. Expression normalized to Hnrpab . n = 1 per timepoint. (K) IL-6 measured via ELISA from media of WT, Akr1a1 KO, and Akr1a1/iNOS DKO RAW264.7 cells unstimulated or stimulated for 48-h. Data represents mean ± SD, n = 3 biological replicates. (L) Median fluorescent intensity (MFI) for the markers, F4/80, CD86, MHCII, and CD206, on WT or Akr1a1 KO RAW264.7 cells with or without 48-h LPS/IFNγ. Data represents mean ± SD, n = 3 biological replicates. Statistics : For panel E-H and K, statistical comparisons were performed using one-way ANOVA with Tukey's post hoc test for multiple comparisons with p-value reported; ns indicates not significant. For panel B, C, and L, statistical comparisons were performed using unpaired two-tailed t -test with p-value reported.

Article Snippet: Primary bone marrow-derived macrophages (BMDMs) were isolated from 8 to 12-week-old male and female wild-type C57BL/6J mice or B6.129P2- Nos2 tm1Lau /J (iNOS KO) mice (Jackson Laboratory).

Techniques: Functional Assay, Activation Assay, Activity Assay, Inhibition, Derivative Assay, Western Blot, Quantitative Proteomics, Molecular Weight, Generated, Standard Deviation, Clone Assay, In Vitro, Incubation, Purification, Control, Labeling, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Redox Biology

Article Title: Stromal cell-derived itaconate promotes endometriosis via macrophage NRF2 and lysosomal pH modulation

doi: 10.1016/j.redox.2026.104101

Figure Lengend Snippet: Itaconate-mediated modulation of macrophage activity alters endometriotic lesion p rogression (A) mRNA expression of IL1B , IL6 , TNFA , and iNOS in peritoneal macrophages from endometriosis (EM) and non-EM patients, as well as non-EM macrophages co-cultured with either nor-ESC or ectopic ec-ESC for 12 h (n = 3/group).(B) Migration of si- Irg1 or NC-treated ectopic ESCs was assessed after co-culture with PBMCs for 48 h. Quantification of migrated cells in five random fields per group are shown (n = 3/group). (C-D) PKH67-labeled human ectopic ESCs pretreated with si- Irg1 or NC were co-cultured with PBMCs for 8 h. Phagocytosis of ESCs by macrophages was analyzed by flow cytometry with representative gating (C), PKH67 signal and quantification of PKH67-positive macrophages (D) (n = 6/group).(E,G) Flow cytometry analysis and quantification of iNOS + peritoneal macrophages from mouse model in E (E), A (G). (n = 6/group). (F,H) mRNA levels of Il1b , Il6 , Nos2 , and Tnf in peritoneal macrophages from mouse model in E (E), A (G) (n = 6/group).(I)Quantification of itaconate in endometriotic lesion tissue by LC–MS from mice treated with IRG1-IN-1 or vehicle. (n = 3/group).(J) Flow cytometry analysis and quantification of iNOS + peritoneal macrophages from mouse model in J (n = 6/group).(K,L) Migration of mESCs induced by peritoneal macrophages from PBS- or 4-OI-treated mice was assessed by transwell assay; representative images and quantification of migrated cells are shown (n = 5/group). (M) Flow cytometry analysis and quantification of phagocytosis of PKH67-labeled mESCs by peritoneal macrophages (n = 6/group).(N) Schematic of the experimental design for clodronate liposome-mediated macrophage depletion and 4-OI intervention in EM mice. (O, P) Representative images (O) and gross morphology (P) of endometriotic lesions in control, clodronate, PBS, and 4-OI groups. (Q) Quantification of lesion weight in mice treated with clodronate liposomes or control liposomes, with or without 4-OI (n = 6/group). (Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.) E2, estradiol benzoate; EM, endometriosis; LNP, lipid nanoparticle; LPS, lipopolysaccharide; 4-OI, 4-octyl itaconate; Clod, clodronate liposome; PBS, phosphate-buffered saline; si- Irg1 , small interfering RNA targeting Irg1.

Article Snippet: After blocking in 5% milk in TBST, membranes were incubated overnight at 4 °C with primary antibodies: Beta Actin Monoclonal antibody(1:20000, 66009-1-Ig; Proteintech), Beta Tubulin Recombinant antibody (1:5000, 80713-1-RR; Proteintech), Anti-IRG1 antibody (1:1000; ab222411; abcam; Cambridge, UK), Nrf2 monoclonal antibody (1:2000; A21176; abclonal; Wuhan, China), iNOS Polyclonal antibody (1:500; 22226-1-AP; Proteintech), NOX2 Polyclonal antibody (1:3000; 19013-1-AP; Proteintech), p-p38 MAPK Polyclonal antibody (1:2000; 28796-1-AP; Proteintech), and p38 MAPK Polyclonal antibody (1:4000; 14064-1-AP; Proteintech).

Techniques: Activity Assay, Expressing, Cell Culture, Migration, Co-Culture Assay, Labeling, Flow Cytometry, Liquid Chromatography with Mass Spectroscopy, Transwell Assay, Control, Liposomes, Saline, Small Interfering RNA

Journal: Redox Biology

Article Title: Stromal cell-derived itaconate promotes endometriosis via macrophage NRF2 and lysosomal pH modulation

doi: 10.1016/j.redox.2026.104101

Figure Lengend Snippet: Inhibition of NRF2 Signaling Reverses the Anti-inflammatory and Anti-endometriotic Effects of Itaconate in Macrophages and a Mouse Model of End ometriosis (A,B) Western blot analysis (A) and quantification (B) of NRF2 protein levels in bone marrow-derived macrophages (BMDMs) pretreated with LPS (100 ng/mL), 4-octyl itaconate (4-OI, 250 μM), or both for 12 h (n = 3/group). (C,D) Western blot analysis (C) and quantification (D) of NRF2 in BMDMs treated with LPS, 4-OI, and the NRF2 inhibitor ML385 (2.5 μM) for 12 h (n = 3/group). (E) Flow cytometry analysis and quantification of iNOS + BMDMs after indicated treatments (n = 3/group). (F) mRNA expression of pro-inflammatory genes ( Il1b , Il6 , Nos2 , Tnf ) in BMDMs under different conditions (n = 3/group). (G) Flow cytometry analysis and quantification of iNOS + BMDMs following NRF2 knockdown (si Nrf2 ) with or without 4-OI, compared to negative control (NC) (n = 3/group).(H) Schematic of the experimental design for ML385 administration in a mouse model of endometriosis. (I) Representative images of endometriotic lesions in PBS- and ML385-treated mice (lesions marked by red circles). (J) Gross morphology of lesions in both groups(n = 6/group). (K) Quantification of lesion weight (n = 6/group). (L) Flow cytometry analysis and quantification of iNOS + peritoneal macrophages from EM mice treated with PBS or ML385 (n = 6/group). (M) mRNA expression of Il1b , Il6 , Nos2 , and Tnf in peritoneal macrophages from each group (n = 6/group). (Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.) E2, estradiol benzoate; EM, endometriosis; LPS, lipopolysaccharide; 4-OI, 4-octyl itaconate; ML385, NRF2 inhibitor.

Article Snippet: After blocking in 5% milk in TBST, membranes were incubated overnight at 4 °C with primary antibodies: Beta Actin Monoclonal antibody(1:20000, 66009-1-Ig; Proteintech), Beta Tubulin Recombinant antibody (1:5000, 80713-1-RR; Proteintech), Anti-IRG1 antibody (1:1000; ab222411; abcam; Cambridge, UK), Nrf2 monoclonal antibody (1:2000; A21176; abclonal; Wuhan, China), iNOS Polyclonal antibody (1:500; 22226-1-AP; Proteintech), NOX2 Polyclonal antibody (1:3000; 19013-1-AP; Proteintech), p-p38 MAPK Polyclonal antibody (1:2000; 28796-1-AP; Proteintech), and p38 MAPK Polyclonal antibody (1:4000; 14064-1-AP; Proteintech).

Techniques: Inhibition, Western Blot, Derivative Assay, Flow Cytometry, Expressing, Knockdown, Negative Control

Journal: Redox Biology

Article Title: Stromal cell-derived itaconate promotes endometriosis via macrophage NRF2 and lysosomal pH modulation

doi: 10.1016/j.redox.2026.104101

Figure Lengend Snippet: Itaconate Regulates Lysosomal Function, Calcium Signaling, and p38 MAPK Pathway in Macrophages (A) Lysosomal acidification in ascites-derived macrophages from non-endometriosis (NC) and endometriosis (EM) patients assessed by LysoSensor fluorescence intensity (n = 6/group). (B) Lysosomal acidification in PBMC-derived macrophages after LPS,IL-4 and 4-OI treatment (n = 3/group). (C) Lysosomal pH value of BMDMs after LPS and 4-OI treatment (n = 3/group). (D, E) Effect of chloroquine (CQ) on LysoSensor intensity (D) and proportion of iNOS + BMDMs (E) after LPS and 4-OI treatment (n = 3/group). (F) mRNA levels of Il1b , Il6 , Nos2 , and Tnf in BMDMs under indicated treatments (n = 3/group). (G) Intracellular Ca 2+ dynamics in BMDMs after treatments, measured by Fluo-4. (H) Intracellular Ca 2+ dynamics in ascites-derived macrophages from NC and EM patients. (I) Quantification of intracellular calcium in peritoneal macrophages from NC and EM patients (n = 3/group). (J) Quantification of intracellular calcium in BMDMs (n = 5/group). (K) mRNA expression of lysosomal calcium channel genes ( MCOLN1 , MCOLN2 , TPC1 , TPC2 ) in ascites-derived macrophages from NC and EM patients (n = 3/group). (L) lysosomal calcium channel genes mRNA in PBMC-derived macrophages co-cultured with normal ESCs (Nor-ESC), eutopic ESCs (Eu-ESC), or ectopic ESCs (Ec-ESC) from patients (n = 5/group). (M − N) Fluo-4-based Ca 2+ dynamics in BMDMs treated with LPS, 4-OI, ML-SA1 (M), or CQ (N). (O) Flow cytometry analysis and quantification of iNOS + BMDMs after indicated treatments (n = 3/group). (P) mRNA levels of pro-inflammatory genes in BMDMs under different treatments (n = 3/group). (Q) Western blot and quantification of p-p38 and total p38 in BMDMs with LPS ± 4-OI (n = 3/group). (R–S) Western blot and quantification of iNOS, p-p38, and total p38 in BMDMs treated with LPS, 4-OI, and CQ (R), or ML-SA1 (S) (n = 3/group). (Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.) EM, endometriosis; NC, non-EM control; LPS, lipopolysaccharide; 4-OI, 4-octyl itaconate; CQ, chloroquine; ML-SA1, MCOLN channel agonist; PBMC, peripheral blood mononuclear cell; ESC, endometrial stromal cell.

Article Snippet: After blocking in 5% milk in TBST, membranes were incubated overnight at 4 °C with primary antibodies: Beta Actin Monoclonal antibody(1:20000, 66009-1-Ig; Proteintech), Beta Tubulin Recombinant antibody (1:5000, 80713-1-RR; Proteintech), Anti-IRG1 antibody (1:1000; ab222411; abcam; Cambridge, UK), Nrf2 monoclonal antibody (1:2000; A21176; abclonal; Wuhan, China), iNOS Polyclonal antibody (1:500; 22226-1-AP; Proteintech), NOX2 Polyclonal antibody (1:3000; 19013-1-AP; Proteintech), p-p38 MAPK Polyclonal antibody (1:2000; 28796-1-AP; Proteintech), and p38 MAPK Polyclonal antibody (1:4000; 14064-1-AP; Proteintech).

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

Journal: Redox Biology

Article Title: Stromal cell-derived itaconate promotes endometriosis via macrophage NRF2 and lysosomal pH modulation

doi: 10.1016/j.redox.2026.104101

Figure Lengend Snippet: Itaconate Suppresses NOX2-Derived ROS to Regulate Macrophage Function and Lesion Progression in End ometriosis (A-B) qPCR analysis of NOX2 mRNA in peritoneal macrophages (PMs) from human (A) and mouse (B) NC and EM groups (n = 3/group). (C-D) Western blot and quantification of NOX2 protein in PBMC-derived macrophages treated with LPS or LPS + 4-OI (n = 5/group). (E) NOX2 enzyme activity in BMDMs (n = 4/group). (F, G) Flow cytometry and quantification of ROS production in BMDMs after LPS or LPS + 4-OI (n = 3/group). (H, I) Flow cytometry and quantification of iNOS + BMDMs after LPS, LPS + 4-OI, or LPS + 4-OI + DPI treatment (n = 4/group). (J) Intracellular Ca 2+ dynamics in BMDMs measured by Fluo-4 after LPS, 4-OI, or DPI treatment. (K) Schematic of 4-OI and DPI intervention in the mouse endometriosis model. (L, M) Representative images (L) and gross morphology (M) of endometriotic lesions after PBS, 4-OI, or DPI treatment. (N) Quantification of lesion weight (n = 6/group). (O, P) Flow cytometry and quantification of iNOS + peritoneal macrophages (percentage and MFI) in peritoneal lavage (n = 6/group). (Q) mRNA levels of Il1b , Il6 , Nos2 , and Tnf in peritoneal macrophages after treatments (n = 6/group). (Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, ns: not significant.) E2, estradiol benzoate; EM, endometriosis; NC, non-EM control; LPS, lipopolysaccharide; 4-OI, 4-octyl itaconate; DPI, NOX2 inhibitor.

Article Snippet: After blocking in 5% milk in TBST, membranes were incubated overnight at 4 °C with primary antibodies: Beta Actin Monoclonal antibody(1:20000, 66009-1-Ig; Proteintech), Beta Tubulin Recombinant antibody (1:5000, 80713-1-RR; Proteintech), Anti-IRG1 antibody (1:1000; ab222411; abcam; Cambridge, UK), Nrf2 monoclonal antibody (1:2000; A21176; abclonal; Wuhan, China), iNOS Polyclonal antibody (1:500; 22226-1-AP; Proteintech), NOX2 Polyclonal antibody (1:3000; 19013-1-AP; Proteintech), p-p38 MAPK Polyclonal antibody (1:2000; 28796-1-AP; Proteintech), and p38 MAPK Polyclonal antibody (1:4000; 14064-1-AP; Proteintech).

Techniques: Derivative Assay, Western Blot, Activity Assay, Flow Cytometry, 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: 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