Journal: Clinical and Experimental Pharmacology & Physiology

Article Title: Mechanisms of IL‐17A Neutralisation in Alleviating Renal Fibrosis and Inflammation in Spontaneously Hypertensive Rats

doi: 10.1111/1440-1681.70116

Figure Lengend Snippet: Effects of IL‐17A neutralisation on macrophage polarisation in SHR renal tissues. (A) Representative IHC staining of M1 macrophage markers (iNOS and CD86) with quantitative analysis of their positive areas. (B) Representative IHC staining of M2 macrophage markers (Arg‐1 and CD163) with quantitative analysis of their positive areas. (C) Representative immunoblots and relative expression levels of iNOS, CD86, Arg‐1, and CD163 proteins. (D) Proportion of CD86 + and CD163 + cells among CD68 + macrophages. (E) mRNA expression levels of iNOS, CD86, Arg‐1, and CD163. Data are presented as mean ± SD ( n = 6).

Article Snippet: Sections were then incubated overnight at 4°C with primary antibodies against: E‐cadherin (Boster, China), Collagen III (Boster, China), inducible nitric oxide synthase (iNOS) (Boster, China), CD86 (Boster, China), arginase‐1 (Arg‐1) (Boster, China), CD163 (Boster, China), α‐smooth muscle actin (α‐SMA) (Cell Signalling Technology, USA).

Techniques: Immunohistochemistry, Western Blot, Expressing

Journal: Neurochemistry international

Article Title: Cannabinoid-profiled agents improve cell survival via reduction of oxidative stress and inflammation, and Nrf2 activation in a toxic model combining hyperglycemia+Aβ 1-42 peptide in rat hippocampal neurons.

doi: 10.1016/j.neuint.2020.104817

Figure Lengend Snippet: Fig. 13. Effects of increased concentrations (1–1000 nM, for 6 h) of anandamide (AEA), 2-arachidonoylglycerol (2-AG), CP 55–940, WIN 55,212–2, and URB597, on inducible-nitric oxide synthase (iNOS; A), Interleukin-1β (IL-1β; B), and Tumor Necrosis Factor-α (TNF-α; C) levels in primary hippocampal neurons exposed to high glucose (GLU; 150 mM for 48 h) + amyloid β peptide (Aβ1-42; 500 nM for 24 h). Data are mean values ± S.E.M. of n = 2 experiments per assay. **P ≤0.01 and ʄP ≤ 0.001, different from the control group; +P ≤0.05, ++P ≤0.01 and ɸP ≤0.001, different from the GLU + Aβ1-42 group; Kruskal-Wallis ANOVA with uncorrected Dunn’s test.

Article Snippet: Rat Inducible Nitric Oxide Synthase (iNOS) ELISA kit and Rat 3-nitrotyrosine (3-NT) ELISA kit were obtained from Cusabio Technology LLC (Houston, TX, USA).

Techniques: 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

Journal: Nutrients

Article Title: Development of Colorectal-Targeted Dietary Supplement Tablets Containing Natural Purple Rice Bran Oil as a Colorectal Chemopreventive

doi: 10.3390/nu10040444

Figure Lengend Snippet: Anti-inflammatory activities of NPRBOs.

Article Snippet: Moreover, a commercially available mouse iNOS ELISA kit (CSB-E08326M, Cusabio Biotech, Co., Ltd., Houston, TX, USA) was used to measure the production of iNOS in the cell lysates.

Techniques: Inhibition, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: The ER stress sensor inositol-requiring enzyme 1α in Kupffer cells promotes hepatic ischemia-reperfusion injury

doi: 10.1016/j.jbc.2021.101532

Figure Lengend Snippet: IRE1α deficiency diminishes iNos expression in Kupffer cells. A–C, control or MφKO mice were subjected to sham or hepatic warm ischemia/reperfusion (n=3–4 per group). A, immunoblot analysis of iNos protein in liver lysates. β-Actin was used as the loading control. B, qRT-PCR analysis of the mRNA abundance of Nos2 in isolated Kupffer cells. C, immunoblot analysis of iNos protein in freshly isolated Kupffer cells. HSP90 was used as the loading control. The relative levels of iNos protein were quantified. D–G, Kupffer cells were isolated from control and MφKO mice. Cells were then treated with 100 ng/ml LPS for the indicated time intervals ( D and E ), or with 0.1 or 1 μg/ml LPS for 12 h ( F and G ). D and F, immunoblot analysis of IRE1α and iNos protein. E and G, qRT-PCR analysis of the mRNA abundance of Xbp1s and Nos2 . H, primary Kupffer cells were treated with PBS (veh) or 100 ng/ml LPS with or without 10 μM 4μ8C. Immunoblot analysis of the indicated proteins. The relative levels of iNos protein were quantified. All data represent the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 by one way or two-way ANOVA.

Article Snippet: For iNOS inhibition, mice were injected with 1400W (35 mg per kg body weight; Targetmol, #T3491) or 10% DMSO, 40% PEG300, 5% Tween 80 in saline as vehicle control at 1 h prior to hepatic I/R surgery.

Techniques: Expressing, Control, Western Blot, Quantitative RT-PCR, Isolation

Journal: The Journal of Biological Chemistry

Article Title: The ER stress sensor inositol-requiring enzyme 1α in Kupffer cells promotes hepatic ischemia-reperfusion injury

doi: 10.1016/j.jbc.2021.101532

Figure Lengend Snippet: Blocking IRE1α RNase attenuates Kupffer-cell-mediated inflammation during hepatic I/R injury. A, immunohistochemical staining of Ly6G + neutrophils in liver sections of the indicated group. Quantification of Ly6G + cells is shown per field. Scale bar, 100 μm. B, ELISA analysis of serum levels of the indicated inflammatory cytokines. C and D, qRT-PCR analysis of the mRNA abundance of the indicated UPR ( C ) or inflammatory genes ( D ) in freshly isolated Kupffer cells. E, immunoblot analysis of the indicated proteins in lysates of freshly isolated Kupffer cells. HSP90 was used as the loading control. The relative levels of NLRP3, c-Casp1 and iNos proteins were quantified. All data represent the mean ± SD. ∗ p < 0.05 and ∗∗ p < 0.01 by one-way ANOVA. NLRP3, nucleotide-binding domain, leucine-rich repeat containing protein 3; UPR, unfolded protein response.

Article Snippet: For iNOS inhibition, mice were injected with 1400W (35 mg per kg body weight; Targetmol, #T3491) or 10% DMSO, 40% PEG300, 5% Tween 80 in saline as vehicle control at 1 h prior to hepatic I/R surgery.

Techniques: Blocking Assay, Immunohistochemical staining, Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Isolation, Western Blot, Control, Binding Assay

Journal: Journal for immunotherapy of cancer

Article Title: Inhibiting CMTM4 reverses the immunosuppressive function of myeloid-derived suppressor cells and augments immunotherapy response in cervical cancer.

doi: 10.1136/jitc-2025-011776

Figure Lengend Snippet: Figure 2 CMTM4 increases MDSCs infiltration and inhibits CD8+ T cell accumulation in CC. (A–B) Flow cytometric anaysis of MDSCs (A), G-MDSC or M-MDSC (B) in TC1-C4KO and TC1-C4WT tumor-bearing mice. (C–D) Composition of immune cells in the TC1 spleen (C) and tumor (D) was shown in the heatmap. (E–F) Flow cytometric analysis of the ability of MDSC, G- MDSC, and M-MDSC to secrete INOS (E) or Arg-1 (F). INOS and Arg-1 expressions were described by percentage and mean fluorescence intensity (MFI). (G) Flow cytometric analysis of the infiltration of IFN-γ and GzmB CD8+ T cells. (H) Correlation between tumor weight with tumorous MDSCs abundance, tumor-infiltrating CD8+ T cells IFN-γ level in TC1-C4KO and TC1- C4WT tumor-bearing mice model (All n=6). (I) Flow cytometric analysis of PMN-MDSC and M-MDSC in PBMCs isolated from patients with benign tumors (n=21) and cervical cancer (n=18). (J) Correlation analysis of S100A8 or INOS expression and CMTM4 expression in clinical samples. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Arg-1, arginase-1; CC, cervical cancer; CKLF, chemokine-like factor; CMTM, CKLF-like MARVEL transmembrane domain-containing family; DC, dendritic cell; G- MDSC, granulocyte-like MDSC; GzmB, granzyme B; IFN, interferon; iNOS, inducible nitric oxide synthase; KO, knockout; MDSC, myeloid-derived suppressor cell; M-MDSC, monocyte-like MDSC; mRNA, messenger RNA; NK, natural killer; PMN- MDSC, polymorphonuclear myeloid-derived suppressor cell; Th1, T helper type 1 cell; Treg, regulatory T cell; WT, wild type.

Article Snippet: Human Arg- 1 or INOS protein levels in culture supernatants, and serum samples were measured using the Human Arg- 1 ELISA Kit (catalog no. RK07687, Abclonal) or Human INOS ELISA Kit (catalog no. RK01945, Abclonal).

Techniques: Fluorescence, Isolation, Expressing, Knock-Out, Derivative Assay

Journal: Journal for immunotherapy of cancer

Article Title: Inhibiting CMTM4 reverses the immunosuppressive function of myeloid-derived suppressor cells and augments immunotherapy response in cervical cancer.

doi: 10.1136/jitc-2025-011776

Figure Lengend Snippet: Figure 3 Knockdown of CMTM4 results in the dysregulation of immune-associated cytokines and chemokines. (A–B) CM collected from CMTM4 KO-HeLa and CMTM4 KO-SiHa or from control were placed in the lower chambers. Freshly isolated PBMCs MDSCs were seeded in the upper chambers and allowed to transwell for 24 hours. Total numbers of transwelled MDSCs found in the lower chambers were counted. Data are presented as the mean±SEM (n=3). (C–D) CM collected from CMTM4 KO-HeLa and CMTM4 KO-SiHa or control was cocultured with mononuclear cells from cord blood for 24 hours. The proportion of MDSCs was detected by flow cytometry (n=3). (E) After treating umbilical cord blood mononuclear cells with CM from CMTM4 KO HeLa and CMTM4 KO SiHa or the control group for 24 hours, the coculture supernatant was collected and the secretion of Arg-1 or INOS was detected by ELISA. (F) Heatmap of 43 different human cytokine expression levels with a concentration greater than 100 pg/mL in CMTM4 KO-HeLa and CMTM4 NC-HeLa using Luminex-MultiDTX-43-Human. (G–H) RT-qPCR and ELISA assays analyze expression of IL-6 and CCL2 in CMTM4 KO-HeLa and CMTM4 KO-SiHa (n=3). (I–J) ELISA analysis of CCL2 and IL-6 expression in the serum of patients with benign tumors (CCL2 n=17; IL-6 n=10) and cervical cancer (CCL2 n=11; IL-6 n=18). (K–L) Correlation analysis of PMN-MDSC or M-MDSC expression and CCL2 or IL-6 expression in clinical samples. (M) GEPIA overall survival curves analysis of patients with CC with high or low expression of CCL2 or IL-6. N. Tumor mRNA level and serum protein concentration of CCL2 and IL-6 in TC1 tumor-bearing mice models (n=6). (O) Correlation analysis of CCL2 or IL-6 mRNA expression and CMTM4 mRNA expression in clinical cervical tissue. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Arg-1, arginase-1; CCL, C-C motif chemokine ligand; CM, conditioned medium; CKLF, chemokine-like factor; CMTM, CKLF-like MARVEL transmembrane domain-containing family; CXCL, C-X-C motif chemokine ligand; GEPIA, Gene Expression Profiling Interactive Analysis; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; KO, knockout; MDSC, myeloid-derived suppressor cell; mRNA, messenger RNA; PB, peripheral blood; PBMCs, peripheral blood mononuclear cells; PDGF-AA, platelet-derived growth factor-AA; PD-L1, programmed death-ligand 1; RT-qPCR, real-time quantitative PCR; Th1, T helper type 1 cell; TPM, transcripts per million; WT, wild type.

Article Snippet: Human Arg- 1 or INOS protein levels in culture supernatants, and serum samples were measured using the Human Arg- 1 ELISA Kit (catalog no. RK07687, Abclonal) or Human INOS ELISA Kit (catalog no. RK01945, Abclonal).

Techniques: Knockdown, Control, Isolation, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Concentration Assay, Luminex, Quantitative RT-PCR, Protein Concentration, Gene Expression, Knock-Out, Derivative Assay, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Heading towards a dead end: The role of DND1 in germ line differentiation of human iPSCs

doi: 10.1371/journal.pone.0258427

Figure Lengend Snippet: Antibodies used for flow cytometry.

Article Snippet: NANOG , REA314 , PE , Recombinant human IgG1 , anti-human , 130-117-526 , Miltenyi Biotec.

Techniques: Cytometry, Conjugation Assay, Recombinant, Control