Journal: iScience

Article Title: ACSL1 is a key regulator of inflammatory and macrophage foaming induced by short-term palmitate exposure or acute high-fat feeding

doi: 10.1016/j.isci.2023.107145

Figure Lengend Snippet:

Article Snippet: FITC Anti-mouse-Ly-6C Antibody , Miltenyi Biotec , 130-111-777.

Techniques: Control, Recombinant, Reverse Transcription, Gene Expression, Activity Assay, Software

Journal: Advanced Science

Article Title: The KDM6B/SLC10A2 Axis Suppresses MDSCs Recruitment via ERK/AP‐1 Signaling in Colorectal Cancer

doi: 10.1002/advs.202514086

Figure Lengend Snippet: KDM6B deficiency promotes MDSCs‐mediated immunosuppression via the AP‐1/CXCL–CXCR2 axis. A) Immunohistochemical (IHC) staining of Gr1 and CXCR2 in colon tissues from Villin Cre ; KDM6B fl/fl mice and KDM6B fl/fl littermate controls after 10 weeks of AOM/DSS treatment. Quantification of positive pixels per field. B) qPCR analysis of the expression of MDSC markers (S100A8, S100A9, Arg1, TGF‐β, and VEGF‐a) in the intestinal epithelium of Villin Cre ; KDM6B fl/fl mice and KDM6B fl/fl mice. C,D) MC38 murine colon cancer cells stably overexpressing KDM6B (OE‐KDM6B) or empty vector control (MCS) were subcutaneously injected into C57BL/6 mice (n = 5 per group). (C) Immunohistochemical (IHC) staining of Gr1 and CXCR2 in subcutaneous tumors derived from MC38‐MCS and MC38‐OE‐KDM6B cells at the endpoint (14 days post‐injection). Representative images are shown. Scale bars: 200 µm. D) qPCR of the expression of MDSCs markers (S100A8, S100A9, Arg1, TGF‐β, and VEGF‐a) in subcutaneous tumors derived from MC38‐MCS and MC38‐OE‐KDM6B cells at the endpoint. E) Transwell migration assays were performed to evaluate the ability of tumor‐conditioned media (TCM) from MC38 cells, including the MC38‐vector (MCS), MC38‐OE‐KDM6B (OE‐KDM6B), MC38‐shNC (NC), and MC38‐shKDM6B (sh‐KDM6B) groups, to recruit MDSCs. Representative images are shown. Each group had three biological replicates. F) Quantification of migrated MDSCs. G,H) qPCR of the expression of MDSC markers (S100A8, S100A9, Arg1, TGF‐β, and VEGF‐a) after TCM treatment. I,J) CXCR2 blockade experiments: (I) A CXCR2 inhibitor (SB265610, 10 µM) was added into tumor‐conditioned media (TCM) from MC38‐NC cells or MC38‐sh‐KDM6B cells, and Transwell assays were performed to evaluate the migration capacity of MDSCs. Representative images are shown. Each group had three biological replicates. (J) The statistical results of the migration cell count. K–M) In vivo CXCR2 inhibition: (K) Subcutaneous tumors were generated in C57BL/6 mice by injection of MC38 cells with stable KDM6B knockdown (sh‐KDM6B) or negative control (NC). Mice were treated daily with the CXCR2 inhibitor SB265610 (2 mg kg −1 day −1 , i.p.) or vehicle control for 15 days (n = 5). (L) Tumor growth curves. (M) Endpoint tumor weights. (N) Flow cytometry of tumor‐infiltrating PMN‐MDSCs (CD11b + Ly6G + Ly6C − ), CD4 + T cells (CD3 + CD4 + ) and CD8 + T cells (CD3 + CD8 + ). (O) Flow cytometry of peripheral blood from the four experimental groups (NC, NC+CXCR2i, sh‐KDM6B, and sh‐KDM6B+CXCR2i) was performed at the endpoint (Day 15). Gating strategy for PMN‐MDSCs (CD11b⁺Ly6G⁺Ly6C − ), CD4 + T cells (CD3 + CD4 + ) and CD8 + T cells (CD3 + CD8 + ). (P) Quantification of MDSCs and T cells in tumors and peripheral blood. (Q) Immunohistochemical (IHC) analysis of CXCR2 and Gr1⁺ cell infiltration in subcutaneous tumors. The sample size for each group was n = 5. The middle line in the box plots represents the median, whereas the whiskers denote the minimum‐to‐maximum range of the data distribution. The error bars show the means ± SEM. Statistical significance was determined by an unpaired, two‐tailed Student's t test. * p < 0.05, ** p < 0.01.

Article Snippet: Antibodies against the following proteins were used: CD45 (E‐AB‐F1136E; Elabscience Biotechnology, Wuhan, China), CD11b (E‐AB‐F1081F; Elabscience), Ly6G (E‐AB‐F1108D; Elabscience), Ly6C (E‐AB‐F1121H; Elabscience), CD3 (E‐AB‐F1013C; Elabscience), CD4 (E‐AB‐F1097F; Elabscience), CD8 (E‐AB‐F1104D; Elabscience), F4/80 (E‐AB‐F0995H; Elabscience), CD86 (E‐AB‐F0994D; Elabscience), and CD206 (E‐AB‐F1135C; Elabscience).

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Stable Transfection, Plasmid Preparation, Control, Injection, Derivative Assay, Migration, Cell Characterization, In Vivo, Inhibition, Generated, Knockdown, Negative Control, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: Histone lactylation increases CXCL1 expression for neutrophil infiltration and immune escape in pancreatic cancer

doi: 10.1038/s41467-026-69311-5

Figure Lengend Snippet: A The expression of neutrophils and CD8 + T cells in low- and high-glycolysis clusters, divided by ssGSEA score of glycolysis-related genes, in TCGA and CPTAC PAAD database. Data are presented as box plots showing the median (center line), the first and third quartiles (box bounds), and the whiskers extend to 1.5 times the interquartile range from the box. B The correlation of glycolysis level and neutrophils or CD8 + T cells expression in TCGA ( n = 179 patients) and CPTAC ( n = 140 patients) PAAD database. C Kaplan–Meier plots representing survival probabilities in TCGA-PAAD patients according to the relative level of glycolysis-related or neutrophil-related gene expression. D The diagram illustrating the targets of 2DG and shRNA in the glycolysis process. E CyTOF analysis of tumor-infiltrating immunocytes in subcutaneous tumor models with or without 2DG treatment: tSNE plots showing 12 meta-clusters based on the expression of 41 markers for the immunocytes. F Bar chart of the frequencies of the immune cell subsets in two experimental groups. G A schematic diagram showing the orthotopic PDAC model ( n = 6 mice per group in one experiment) with or without 2DG treatment. H , I Image and weights of the orthotopic tumors in the experimental groups from ( G ) at the end of the experiments. ( n = 6 mice per group). ( J – M ) Tumor-infiltrating neutrophils, CD8 + T cells, GZMB + CD8 + T cells, and PD-1 + CD8 + T cells isolated from the orthotopic tumors were analyzed using flow cytometry: representative graphs of flow cytometry (left panel) and statistical analysis of the cell ratio (right panel) ( n = 6 mice per group). N A schematic diagram showing the orthotopic tumor model in C57BL/6 J mice ( n = 6 mice per group in one experiment) treated with or without anti-Ly6G/anti-CD8 antibody. O , P Image and weights of the orthotopic tumors in the experimental groups from ( N ) at the end of the experiments ( n = 6 mice per group). Q – S Tumor-infiltrating neutrophils, CD8 + T cells, and GZMB + CD8 + T cells isolated from the orthotopic tumors were analyzed using flow cytometry: Representative graphs of flow cytometry (left panel) and statistical analysis of the cell ratio (right panel) ( n = 6 mice per group). T , U Representative luminescence images of the mouse model in ( N ) and statistical analysis of the total flux ( n = 6 mice per group). Data represent mean ± SEM. Statistical analysis was conducted using the two-tailed unpaired Student’s t test ( A, I–M, P–S, and U ), two-tailed Pearson’s correlation analysis ( B ), and log-rank test ( C ). Source data are provided as a Source Data file.

Article Snippet: Then, the mice were subjected to different treatments as shown in the figures and described as following: (1) Vehicle/2DG treatment was administered (800 mg/kg, i.p., once daily) from day 7 for 9 consecutive days; (2) For neutrophil depletion, these mice received anti-IgG2a (0.25 mg/mouse, i.p.) or anti-Ly6G antibody (0.25 mg/mouse, i.p., BioXCell, #BE0075-1) treatment once a day from day 3 post-implantation.

Techniques: Expressing, Gene Expression, shRNA, Isolation, Flow Cytometry, Two Tailed Test

Journal: mBio

Article Title: Candida -induced granulocytic myeloid-derived suppressor cells are protective against polymicrobial sepsis

doi: 10.1128/mbio.01446-23

Figure Lengend Snippet: Immunized mice control the early proinflammatory response. ( A ) IL-6, ( B ) TNF-α ( C ) PGE 2 , and ( D ) IL-10 levels in the PLF, and ( E ) IL-6 and ( F ) IL-10 levels in the serum of C57BL/6 mice ( n = 4–5 mice/group/timepoint, assayed in duplicate) at the indicated hpc (1.4 × 10 7 CFU/mL C . albicans + 8 × 10 7 CFU/mL S . aureus ). The Prostaglandin E Metabolite (PGEM) Competitive Enzyme Immunoassay was used to measure PGE 2 as a function of its breakdown product. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, two-way ANOVA with Sidak’s multiple comparisons test. ( G ) Cytokine/chemokine levels in the PLF and serum of immunized Swiss Webster mice ( n = 3 mice/group/timepoint; one experiment) following depletion of Gr-1 + cells. Mice were treated with 200 µg of an isotype control or anti-mouse Ly6G/Ly6C (Gr-1) antibody 48 h prior to and 2 hpc (1.75 × 10 7 CFU/mL C . albicans + 8 × 10 7 CFU/mL S . aureus ). Data are presented as Log10 (pg/mL). Hpc, h post-challenge.

Article Snippet: To individually deplete Ly6G + or Ly6C + leukocytes, mice were injected IP with either 200 μg anti-mouse Ly6G antibody (clone 1A8; BE0075-1, Bio X Cell) or anti-mouse Ly6C antibody (clone Monts 1; BE0203, Bio X Cell) in 200 μL of sterile pH 7 dilution buffer; 200 μg rat IgG2a isotype control antibody (clone 2A3; BE0089, Bio X Cell) in 200 μL of sterile pH 6.5 dilution buffer (IP0065, Bio X Cell) was injected IP as a control for both the Ly6G and Ly6C depletion groups.

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: mBio

Article Title: Candida -induced granulocytic myeloid-derived suppressor cells are protective against polymicrobial sepsis

doi: 10.1128/mbio.01446-23

Figure Lengend Snippet: G-MDSCs are increased in immunized mice. M-MDSCs (CD11b + Ly6G - Ly6C hi ) and G-MDSCs (CD11b + Ly6G + Ly6C +/lo ) in the ( A ) bone marrow, ( B ) blood, ( C ) spleen, and ( D ) PLF of C57BL/6 mice ( n = 2–3 mice/group/experiment; two experiments) at the indicated hpc (1.4 × 10 7 CFU/mL C . albicans + 8 × 10 7 CFU/mL S . aureus ). Left, representative gates; middle, M- and G-MDSCs expressed as percentage of live CD11b + cells; right, M- and G-MDSCs expressed as cells/mL. * P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA with Sidak’s multiple comparisons test. Representative full gating strategies are illustrated in . Hpc, h post-challenge.

Article Snippet: To individually deplete Ly6G + or Ly6C + leukocytes, mice were injected IP with either 200 μg anti-mouse Ly6G antibody (clone 1A8; BE0075-1, Bio X Cell) or anti-mouse Ly6C antibody (clone Monts 1; BE0203, Bio X Cell) in 200 μL of sterile pH 7 dilution buffer; 200 μg rat IgG2a isotype control antibody (clone 2A3; BE0089, Bio X Cell) in 200 μL of sterile pH 6.5 dilution buffer (IP0065, Bio X Cell) was injected IP as a control for both the Ly6G and Ly6C depletion groups.

Techniques:

Journal: mBio

Article Title: Candida -induced granulocytic myeloid-derived suppressor cells are protective against polymicrobial sepsis

doi: 10.1128/mbio.01446-23

Figure Lengend Snippet: Ly6G + G-MDSCs are required for protection. ( A ) Experimental design: WT Swiss Webster mice were immunized with C. dubliniensis , followed by lethal sepsis challenge (2.1 × 10 7 CFU/mL C . albicans + 8 × 10 7 CFU/mL S . aureus ) 14 d later. To deplete Ly6G + or Ly6C + leukocytes, mice were injected IP with anti-Ly6G antibody, anti-Ly6C antibody, or isotype control antibody (200 µg) 48 h prior to and 2 h after lethal sepsis challenge, followed by every 2 d thereafter for the duration of the study. ( B ) Survival following lethal sepsis challenge ( n = 9–10 mice/group/experiment; two experiments). α, P < 0.001 vs Nonimmunized control (dashed black line vs solid black line); β, ns vs Ly6C depletion (gray line vs blue line); γ, P = 0.009 vs Isotype control (pink line vs gray line); Log-rank test with Bonferroni correction. ( C and D ) Microbial burden in the ( C ) spleen and ( D ) PLF 2 dpc ( n = 4–5 mice/group; one experiment). Dpc, d post-challenge.

Article Snippet: To individually deplete Ly6G + or Ly6C + leukocytes, mice were injected IP with either 200 μg anti-mouse Ly6G antibody (clone 1A8; BE0075-1, Bio X Cell) or anti-mouse Ly6C antibody (clone Monts 1; BE0203, Bio X Cell) in 200 μL of sterile pH 7 dilution buffer; 200 μg rat IgG2a isotype control antibody (clone 2A3; BE0089, Bio X Cell) in 200 μL of sterile pH 6.5 dilution buffer (IP0065, Bio X Cell) was injected IP as a control for both the Ly6G and Ly6C depletion groups.

Techniques: Injection, Control

Journal: mBio

Article Title: Candida -induced granulocytic myeloid-derived suppressor cells are protective against polymicrobial sepsis

doi: 10.1128/mbio.01446-23

Figure Lengend Snippet: Flow cytometry antibodies used in this study

Article Snippet: To individually deplete Ly6G + or Ly6C + leukocytes, mice were injected IP with either 200 μg anti-mouse Ly6G antibody (clone 1A8; BE0075-1, Bio X Cell) or anti-mouse Ly6C antibody (clone Monts 1; BE0203, Bio X Cell) in 200 μL of sterile pH 7 dilution buffer; 200 μg rat IgG2a isotype control antibody (clone 2A3; BE0089, Bio X Cell) in 200 μL of sterile pH 6.5 dilution buffer (IP0065, Bio X Cell) was injected IP as a control for both the Ly6G and Ly6C depletion groups.

Techniques: Flow Cytometry, Blocking Assay

Journal: Cell Death & Disease

Article Title: Expansion of monocytic myeloid-derived suppressor cells ameliorated intestinal inflammatory response by radiation through SOCS3 expression

doi: 10.1038/s41419-021-04103-x

Figure Lengend Snippet: Mice were exposed to 7.5 Gy for 1 week to induce intestinal inflammation. A Photomicrographs of colon sections, histological grades, colon lengths, body weights, and survival rates for mice 21 days after irradiation. Scale bar = 100 μm B Representative dot plots of the gating strategy used to identify CD11b + /GR1 + cells from the spleens and intestines of mice 21 days after irradiation. C Representative immunofluorescence images of spleen and intestine stained for CD11b (green), GR1 (red), and CD11b + /GR1 + (yellow) cells. DAPI staining (blue) was used to determine the number of nuclei. Scale bar = 100 μm D CFSE-labeled CD3 + T cells were co‑cultured with splenic MDSCs and stimulated using anti-CD3 and anti-CD28 monoclonal antibodies. The cells were harvested and analyzed after 7 days. CFSE dilution demonstrated that T-cell proliferation was inhibited by MDSCs (left) and that this inhibition occurred in a dose-dependent manner (right). E The two MDSC subtypes, namely gMDSCs (CD11b + , Ly6G + , and Ly6C low ) and mMDSCs (CD11b + , Ly6G - , and Ly6C high ), were identified and analyzed by flow cytometry from spleen and intestine samples. The data are represented as the mean ± SD (n = 8). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control. IR irradiation.

Article Snippet: The tissue sections were incubated with primary antibodies against CD11b (CST, 17800, 1:100 dilution, IL6 (CST, 12912, 1:100 dilution), IL10 (Abcam, ab192271, 1:100 dilution), and SOCS3 (Abcam, ab16030, 1:100 dilution), phospho-STAT3 (CST, 9145, 1:100 dilution), and GR1 (R&D Systems, MN, USA, MAB1037-100, 1:100 dilution) overnight at 4 °C.

Techniques: Irradiation, Immunofluorescence, Staining, Labeling, Bioprocessing, Inhibition, Flow Cytometry, Control

Journal: Cell Death & Disease

Article Title: Expansion of monocytic myeloid-derived suppressor cells ameliorated intestinal inflammatory response by radiation through SOCS3 expression

doi: 10.1038/s41419-021-04103-x

Figure Lengend Snippet: Mice were exposed to either 0.5 Gy or 5 Gy total body irradiation and analyzed seven days later. Representative dot plots of the gating strategies. A The population of CD11b + /GR1 + cells. B gMDSCs (CD11b + , Ly6G + , and Ly6C low ) and mMDSCs (CD11b + , Ly6G - , and Ly6C high ) from the spleens and intestines of irradiated mice were identified and analyzed by flow cytometry. C Representative dot plots of the gating strategy used to identify IL10 + cells. IL10 + cells were gated and the proportions of CD11b + /GR1 + cells. These cells were gated on gMDSCs (CD11b + , Ly6G + , and Ly6C low ) and mMDSCs (CD11b + , Ly6G − , and Ly6C high ) cells were evaluated from mouse spleens and intestines. D Representative immunofluorescence images of cells from spleen stained for GR1 (green), IL10 (red), and GR1/IL10 (yellow). DAPI staining (blue) was used to determine the number of nuclei. Scale bar = 100 μm. The data are represented as mean ± SD ( n = 8). ** P < 0.01, *** P < 0.001 vs. control.

Article Snippet: The tissue sections were incubated with primary antibodies against CD11b (CST, 17800, 1:100 dilution, IL6 (CST, 12912, 1:100 dilution), IL10 (Abcam, ab192271, 1:100 dilution), and SOCS3 (Abcam, ab16030, 1:100 dilution), phospho-STAT3 (CST, 9145, 1:100 dilution), and GR1 (R&D Systems, MN, USA, MAB1037-100, 1:100 dilution) overnight at 4 °C.

Techniques: Irradiation, Flow Cytometry, Immunofluorescence, Staining, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cathelicidin-Ka, the first frog-derived TLR2 and TLR4 agonist, induces macrophage activation and promotes inflammation

doi: 10.1007/s00018-025-06068-y

Figure Lengend Snippet: Cath-Ka induces peritoneal cell activation in mice. The peripheral blood and peritoneal fluid of mice injected with Cath-Ka (5 mg/kg, i.p. ) were collected at 2 and 4 h to measure chemotaxis, phagocytosis, and bacterial killing ability of the peritoneal fluid cells and cytokine contents in the peritoneal fluid. ( A ) Representative images (left) and statistical analysis (right) of peritoneal cells. Scale bar = 400 μm. ( B ) Quantification of cytokines in peritoneal lavage fluids. ( C , D ) Colony formation (left) and statistical analysis (right) of E. coli engulfed by peritoneal cells in bacterial phagocytosis and intracellular killing assays. Peritoneal fluid cells were infected with E. coli at a MOI of 10 for 1 h ( C ) or another 2 h in the presence of gentamicin ( D ) before the numbers of live bacteria in cells were counted by colony formation. ( E-H ) Representative flow cytometry images ( E , F ) and statistical analysis ( G , H ) of leukocytes, myeloid cells, monocytes, inflammatory macrophages, and neutrophils. Peritoneal fluid ( E , G ) and peripheral blood cells ( F , H ) were stained by anti-CD45, CD11b, Ly6G, Ly6C, and F4/80 antibodies for 30 min before flow cytometry analysis. Data are expressed as mean ± SD ( n = 6–10). * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant

Article Snippet: AF488 Anti-Mouse Ly6C (#E-AB-F1121UL) was from Elabscience (Wuhan, China), and PE-anti-mouse CD11b (#101207), APC-anti-mouse CD206 (#141707), and PerCP/ Cyanine5.5-anti-mouse F4/80 (#123128) were from Biolegend (San Diego, CA, USA).

Techniques: Activation Assay, Injection, Chemotaxis Assay, Infection, Bacteria, Flow Cytometry, Staining