Journal: Cancer biology & therapy

Article Title: circ_C20orf11 enhances DDP resistance by inhibiting miR-527/YWHAZ through the promotion of extracellular vesicle-mediated macrophage M2 polarization in ovarian cancer.

doi: 10.1080/15384047.2021.1959792

Figure Lengend Snippet: Figure 5. EVs mediate M2-type macrophage polarization in ovarian cancer. (a) EVs were isolated from the supernatant of SKOV3/DDP cells and characterized via electron microscopy and (b) nanoparticle tracking. (C) EV-specific markers (CD9, TSG101 and CD63) were measured via western blotting. (d) EVs were labeled with PKH67 dye and cultured with THP-1 cells. Representative fluorescence images are shown. (e) After SKOV3/DDP and A2780/DDP cells were transfected with adenoviral circ_C20orf11 (si- SKOV3/DDP-Exo+si-C20orf11) or its vector control (si-SKOV3/DDP-Exo+si-NC), the cells were cultured with EVs from SKOV3/DDP cells. IL-10 expression was detected using an ELISA. Untreated THP-1 cells served as the control. (f) An ELISA was performed to detect IL-6 expression. (g) Relative mRNA expression of TNF-α, IL-6 and iNOS. (H) mRNA expression of CD206, IL-10 and Arg-1 in relation to GAPDH and U6 expression. n = 3. *P < .05, ** P < .01, *** P < .001.

Article Snippet: After centrifugation (1,000 rpm for 8 min at 4°C), the isolated cells were stained using a CD206 antibody (cat. no. 18704-1-AP, Proteintech) according to the manufacturer’s instructions.

Techniques: Isolation, Electron Microscopy, Western Blot, Labeling, Cell Culture, Fluorescence, Transfection, Plasmid Preparation, Control, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cancer biology & therapy

Article Title: circ_C20orf11 enhances DDP resistance by inhibiting miR-527/YWHAZ through the promotion of extracellular vesicle-mediated macrophage M2 polarization in ovarian cancer.

doi: 10.1080/15384047.2021.1959792

Figure Lengend Snippet: Figure 6. Silencing of circ_C20orf11 enhances sensitivity to DDP in vivo. Xenotransplantation studies were performed with SKOV3 cells. (a) Tumor volume was measured every 5 days for 30 days. (b) Representative images of tumor formation. (c) Tumor weight was measured. (d) qPCR analysis of C20orf11, miR-527 and YWHAZ expression. (e) qPCR results showing CD206, IL-10 and Arg-1 expression. (f) Western blotting detection of YWHAZ PD-L1 is presented. n = 3. *P < .05, ** P < .01, *** P < .001.

Article Snippet: After centrifugation (1,000 rpm for 8 min at 4°C), the isolated cells were stained using a CD206 antibody (cat. no. 18704-1-AP, Proteintech) according to the manufacturer’s instructions.

Techniques: In Vivo, Expressing, Western Blot

Journal: Cancer biology & therapy

Article Title: circ_C20orf11 enhances DDP resistance by inhibiting miR-527/YWHAZ through the promotion of extracellular vesicle-mediated macrophage M2 polarization in ovarian cancer.

doi: 10.1080/15384047.2021.1959792

Figure Lengend Snippet: Figure 7. Serum EV-circ_C20orf11 levels are upregulated in ovarian patients. Patients were considered DDP resistant if they showed no significant clinical effect or had progressive disease after receiving one cycle of DDP treatment. The remaining patients were considered DDP sensitive. Real-time qPCR analysis of (a) C20orf11 and (b) miR-527 expression in relation to GAPDH and U6 expression in DDP-sensitive ovarian cancer tissue (S), DDP-resistant ovarian cancer tissue (R), and healthy ovarian tissue (Normal). (c) Flow cytometry detection and quantification of CD206-positive cells in DDP-sensitive ovarian cancer tissue (S), DDP-resistant ovarian cancer tissue (R), and healthy ovarian tissue (Normal). (d) Real-time qPCR analysis of CD206 expression in relation to GAPDH and U6 expression in DDP-sensitive ovarian cancer tissue (S), DDP- resistant ovarian cancer tissue (R), and healthy ovarian tissue (Normal). (e) The expression level of C20orf11 in ovarian cancer tissue was negatively correlated with that of miR-527. (f) The expression level of miR-527 in ovarian cancer tissue was negatively correlated with that of CD206. (g) Representative image of serum EVs detected using an electron microscope. (h) EVs were measured using nanoparticle tracking. (i) EV markers were analyzed via western blotting. (j) The abundance of C20orf11 in serum EVs was assessed using qPCR. (k) Kaplan-Meier survival curves of patients with ovarian cancer with high and low C20orf11 expression. n = 3. *P < .05, ** P < .01.

Article Snippet: After centrifugation (1,000 rpm for 8 min at 4°C), the isolated cells were stained using a CD206 antibody (cat. no. 18704-1-AP, Proteintech) according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Microscopy, Western Blot

Journal: Molecular Metabolism

Article Title: Indoleamine 2,3-dioxygenase mediates the therapeutic effects of adipose-derived stromal/stem cells in experimental periodontitis by modulating macrophages through the kynurenine-AhR-NRF2 pathway

doi: 10.1016/j.molmet.2022.101617

Figure Lengend Snippet: ASC therapeutic effects on experimental periodontitis in rats. (A) The schematic diagram showed the induction of the experimental rat model of periodontitis for 21 days and ASC injection after the removal of ligatures. (B) Micro-CT imaging and bone parameters (CEJ-ABC distance, BV/TV, Tb. Th, and Tb. Sp) between healthy and experimental rats of periodontitis. Scale bar, 1 mm. (C) Micro-CT imaging displayed the alveolar bone of PBS-injected and ASC-injected rats on day 7, day 14, and day 21 Scale bar, 1 mm. (D) The CEJ-ABC distance and bone parameters (BV/TV, Tb. Th, and Tb. Sp) between the PBS-injected and ASC-injected rats. (E) Representative immunofluorescence staining of M1 phenotype macrophages (iNOS + ; Green) and M2 macrophages (CD206 + ; Red) in sagittal sections of maxillary molars in PBS-injected and ASC-injected rats. Scale bar, 20 μm. (F) The calculated ratio of iNOS + /CD206 + macrophages in PBS-injected and ASC-injected groups. CEJ-ABC, cementoenamel junction and alveolar bone crest. All data were expressed as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: For tissues, the sections were incubated with a mouse anti-CD206 monoclonal antibody (1:100, 60143-1-lg, Proteintech), a rabbit anti-NRF2 monoclonal antibody (1:100, A0674, Abclonal), a rabbit anti-inducible nitric oxide synthase (iNOS) polyclonal antibody (1:100, 18985-1-AP, Proteintech) overnight at 4 °C.

Techniques: Injection, Micro-CT, Imaging, Immunofluorescence, Staining

Journal: Molecular Metabolism

Article Title: Indoleamine 2,3-dioxygenase mediates the therapeutic effects of adipose-derived stromal/stem cells in experimental periodontitis by modulating macrophages through the kynurenine-AhR-NRF2 pathway

doi: 10.1016/j.molmet.2022.101617

Figure Lengend Snippet: ASCs modulated macrophage polarization through NRF2. (A) Representative immunohistochemical staining of NRF2 in the PBS-injected and ASC-injected rats on day 7, day 14, and day 21. Scale bar, 50 μm. (B) Representative immunofluorescence staining of NRF2 (Green) and M2 macrophages (CD206 + ; Red) in sagittal sections of maxillary molars in PBS-injected and ASC-injected rats. Scale bar, 20 μm. (C) Schematic diagram of knocking down NRF2 in macrophages and macrophages cocultured with ASCs and LPS stimulation. (D, E) RT-qPCR and western blotting were used to measure the NRF2 siRNA or siNC in macrophages. (F, G) The mRNA expression and protein levels of M1 markers were increased, while those of M2 markers were decreased after silencing NRF2 in the cocultured groups. R, root; PDL, periodontal ligament; AB, alveolar bone; siRNA, small interfering RNA; NC, negative control. All data were expressed as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: For tissues, the sections were incubated with a mouse anti-CD206 monoclonal antibody (1:100, 60143-1-lg, Proteintech), a rabbit anti-NRF2 monoclonal antibody (1:100, A0674, Abclonal), a rabbit anti-inducible nitric oxide synthase (iNOS) polyclonal antibody (1:100, 18985-1-AP, Proteintech) overnight at 4 °C.

Techniques: Immunohistochemical staining, Staining, Injection, Immunofluorescence, Quantitative RT-PCR, Western Blot, Expressing, Small Interfering RNA, Negative Control

Journal: Molecular Metabolism

Article Title: Indoleamine 2,3-dioxygenase mediates the therapeutic effects of adipose-derived stromal/stem cells in experimental periodontitis by modulating macrophages through the kynurenine-AhR-NRF2 pathway

doi: 10.1016/j.molmet.2022.101617

Figure Lengend Snippet: Inhibition of IDO activity reduced the therapeutic potential of ASCs in experimental periodontitis and downregulated NRF2 expression. (A) Three-dimensional reconstruction of maxillary alveolar bone in ASC-injected and 1-MT pretreated ASC-injected rats on day 7, day 14, and day 21. Scale bar, 1 mm. (B) Analysis of bone parameters. (C) Immunohistochemical staining of IL-1β, OCN, and NRF2 in the ASC-injected and 1-MT pretreated ASC-injected rats. Scale bar, 50 μm. (D) Representative immunofluorescence staining of M1 phenotype macrophages (iNOS + ; Green) and M2 macrophages (CD206 + ; Red) in sagittal sections of maxillary molars in ASC-injected and 1-MT pretreated ASC-injected rats. Scale bar, 20 μm. (E) The mean IOD of IL-1β, OCN, and NRF2 and the calculated ratio of iNOS + /CD206 + . IOD, integrated optical density.

Article Snippet: For tissues, the sections were incubated with a mouse anti-CD206 monoclonal antibody (1:100, 60143-1-lg, Proteintech), a rabbit anti-NRF2 monoclonal antibody (1:100, A0674, Abclonal), a rabbit anti-inducible nitric oxide synthase (iNOS) polyclonal antibody (1:100, 18985-1-AP, Proteintech) overnight at 4 °C.

Techniques: Inhibition, Activity Assay, Expressing, Injection, Immunohistochemical staining, Staining, Immunofluorescence

Journal: Journal of Immunology Research

Article Title: Protective Effects of Thalidomide on High-Glucose-Induced Podocyte Injury through In Vitro Modulation of Macrophage M1/M2 Differentiation

doi: 10.1155/2020/8263598

Figure Lengend Snippet: Effects of thalidomide on iNOS, CD206, Arg-1 and TNF- α protein expression in 33.3 mM glucose-induced macrophage. (a) iNOS and CD206 protein expressions. (d) Arg-1 protein expression. (f) TNF- α protein expression. The results of iNOS, CD206, Arg-1, and TNF- α were represented in (b), (c), (e), and (g), respectively. All results were expressed as a ration with respect to control and represented as the mean ± SD in triplicates. ∗ p < 0.05; versus 11.1 mM glucose. # p < 0.05; versus 33.3 mM glucose. & p < 0.05; versus Tha100. ^ p < 0.05; versus Tha50. Abbreviations: LPS: lipopolysaccharide; Tha50: 50 μ g/ml thalidomide in 33.3 mM glucose; Tha100: 100 μ g/ml thalidomide in 33.3 mM glucose; Tha200: 200 μ g/ml thalidomide in 33.3 mM glucose; iNOS: inducible nitric oxide synthase; CD206: mannose receptor; TNF- α : tumor necrosis factor- α ; Arg-1: arginase-1.

Article Snippet: After several consecutive rinses with a washing buffer (0.1% Tween-20 in PBS), the membranes were incubated with primary antibodies against iNOS at 1 : 500 dilution (Catalog No. BA0362, Boster), TNF- α (Catalog No. BA0131, Boster) at 1 : 500 dilution, CD206 (Catalog No. A02285-2, Boster) at 1 : 500 dilution, and antibody against Arg-1 (Catalog No. BM4000, Boster) at 1 : 500 dilution overnight at 4°C.

Techniques: Expressing, Control

Journal: Journal of Immunology Research

Article Title: Protective Effects of Thalidomide on High-Glucose-Induced Podocyte Injury through In Vitro Modulation of Macrophage M1/M2 Differentiation

doi: 10.1155/2020/8263598

Figure Lengend Snippet: Effects of thalidomide on iNOS, CD206, Arg-1, and TNF- α mRNA expressions in 33.3 mM glucose-induced macrophage. (a) iNOS mRNA expression. (b) CD206 mRNA expression. (c) CD206 mRNA expression. (d) TNF- α mRNA expression.All the results were represented as the mean ± SD in triplicates ∗ p < 0.05; versus 11.1 mM glucose. # p < 0.05; versus 33.3 mM glucose. & p < 0.05; versus Tha100. ^ p < 0.05; versus Tha50. Abbreviations: LPS: lipopolysaccharide; Tha50: 50 μ g/ml thalidomide in 33.3 mM glucose; Tha100: 100 μ g/ml thalidomide in 33.3 mM glucose; Tha200: 200 μ g/ml thalidomide in 33.3 mM glucose; iNOS: inducible nitric oxide synthase; CD206: mannose receptor; TNF- α : tumor necrosis factor- α ; Arg-1: arginase-1.

Article Snippet: After several consecutive rinses with a washing buffer (0.1% Tween-20 in PBS), the membranes were incubated with primary antibodies against iNOS at 1 : 500 dilution (Catalog No. BA0362, Boster), TNF- α (Catalog No. BA0131, Boster) at 1 : 500 dilution, CD206 (Catalog No. A02285-2, Boster) at 1 : 500 dilution, and antibody against Arg-1 (Catalog No. BM4000, Boster) at 1 : 500 dilution overnight at 4°C.

Techniques: Expressing

Journal: bioRxiv

Article Title: Secreted Clever-1 Modulates T Cell Responses and Impacts Cancer Immunotherapy Efficacy

doi: 10.1101/2024.10.23.619796

Figure Lengend Snippet: A, Representative Western blot and quantification of Clever-1 levels in cells and the secreted vesicle fraction after anti-Clever-1 treatment with 9-11 or Bex, or Clever-1 silencing by CRISPR/Cas9 ( STAB1 sg1 and sg2) compared to cells treated with isotype or non-targeting guide RNA (sgRosa26) control, respectively. CD63 was used as loading control and CD206 for M2 polarization control. Clever-1 signal was detected with anti-Clever-1 (4G9) antibody. GAPDH loading control was used for normalizing signal intensity for Clever-1. M0 signal was considered as 1. Student’s t-test (n = 3). B , Schematic of the Jurkat-Raji cell assay and representative reporter activity at different doses of nivolumab in comparison to isotype control treatment. C , Nivolumab-activated Jurkat-Raji cell (no EVs) reporter activity in the presence of EVs collected from primary human macrophages shown in A. Kruskal-Wallis test with Dunn’s multiple comparison. D , Western blot analysis of Clever-1 after siRNA knock-down in KG-1 cells and the effect of EVs (2.5 mg/mL) collected from these cells in regulating CD8 + T cell proliferation. Paired t-test. E , Representative Western blot of the ∼200 kDa sClever-1 band from the plasma vesicle fraction of a patient administered with 0.1 mg/kg of bexmarilimab and quantification at different dose levels (0.1-10 mg/kg, n = 3 in all doses except n = 2 at 10 mg/kg) normalized to CD63 levels. F , Hierarchical Stochastical Neighbor Embedding (hSNE) clusters of mass cytometry data of CD4 + and CD8 + T cells and heatmaps of anti-Clever-1 (9-11 antibody) immunoreactivity during the first treatment cycle with bexmarilimab. Bexmarilimab administration shows significantly decreased binding of 9-11 antibody on CD4 + and CD8 + T cells on day 15 indicating that less sClever-1 is bound to lymphocytes. G , The changes of membrane bound Clever-1 levels in monocytes/macrophages (MoMacs) are shown as reference. Mixed-effects analysis with Holm-Sidak’s multiple comparison test, n = 6-7. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For primary macrophage and EV blots, TFRC NBP2-34602 (Novus, RRID:AB_3289338), CD206 12981S (Cell Signaling), CD63 sc-5275 (Santa Cruz, RRID:AB_627877) were used.

Techniques: Western Blot, CRISPR, Control, Activity Assay, Comparison, Knockdown, Clinical Proteomics, Mass Cytometry, Binding Assay, Membrane