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: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ChemR23, the receptor for chemerin and resolvin E1, is expressed and functional on M1 but not on M2 macrophages.

doi: 10.4049/jimmunol.1402166

Figure Lengend Snippet: FIGURE 5. Repolarization of LPS-stimulated primary human M1 macrophages with RvE1 leads to a distinct macrophage phenotype with properties of proresolution macrophages. LPS-stimulated primary human macrophages were repolarized for 4 d in the presence of medium (med), IL-4, or 10 nM RvE1, and mRNA and protein expression of cytokines and cell surface markers were measured to characterize polarization of macrophages. (A–D) mRNA levels of (A) ChemR23, (B) IL-1b, (C) TNF-a, and (D) IL-10 are presented. All mRNA values were normalized for GAPDH and are presented relative to macrophages repolarized with medium alone (med). (E–G) Membrane expression of (E) ChemR23, (F) CD80, and (G) CD206 is shown, whereas in (H), protein secretion of IL-10 into the supernatant is shown. Bars indicate the mean of three independent experiments. *p , 0.05, **p , 0.01.

Article Snippet: Chemerin Receptor 23, anti-human, mAb (ChemR23 Ab) and isotype IgG3, PE-labeled anti-human CD80, monoclonal allophycocyanin-labeled anti-human CD206 Ab, and the IgG1K isotype control were purchased from R&D Systems Europe.

Techniques: Expressing, Membrane

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: Cells

Article Title: Statins Modulate Microenvironmental Cues Driving Macrophage Polarization in Simulated Periodontal Inflammation

doi: 10.3390/cells12151961

Figure Lengend Snippet: MyD88−dependent modulation of M2 polarization by simvastatin. ( A ) A schematic representation of simvastatin, MyD88 inhibitor treatment, and THP-1-macrophages (M0–M1–M2). ( B ) Cell surface antigen analysis of M2 macrophage markers CD68, CD163, and CD206 was conducted using flow cytometry. Expression levels were normalized using IgG isotype control, and values were expressed in percentage of positive cells (mean ± SD) from three independent preparations. ( C ) RT-PCR analysis and heat maps summarizing TLR and NFkB signaling from M0 (CCL2), M1 (LPS, IFNγ, TNFα), and M2 (IL4, IL10, TGFβ) macrophages. (n = 3) preparations per treatment group, and data found to be significantly different were plotted as heat maps. ( D ) Graphical summary of statin-mediated suppression of TLR and NFkB signaling.

Article Snippet: Phenotyping of macrophages was performed in 1% BSA and 3% human serum PBS according to standard methods using a panel of antibodies targeting CD68 (R and D Systems Cat# IC20401P, Minneapolis, MN, USA, RRID: http://scicrunch.org/resolver/AB_2074835 , accessed on 28 April 2023), CD163 (R and D Systems Cat# FAB1607P, RRID: http://scicrunch.org/resolver/AB_2074536 , accessed on 28 April 2023), and CD206 (R and D Systems Cat# FAB25342P, RRID: http://scicrunch.org/resolver/AB_10889015 , accessed on 28 April 2023); antibodies all from R and D Systems.

Techniques: Flow Cytometry, Expressing, Control, Reverse Transcription Polymerase Chain Reaction

Journal: eLife

Article Title: IRF4 haploinsufficiency in a family with Whipple’s disease

doi: 10.7554/eLife.32340

Figure Lengend Snippet: CD11b, CD86, CD206, CD209 and HLA-DR mean fluorescence intensity (MFI) for M2-MDM (left) and M1-MDM (right) from P1 and two healthy unrelated controls ( C1 and C2 ), either left non-stimulated (NS) or stimulated with IL-4 (for M2-like MDMs) or IFN-g (for M1-like MDMs). We showed that CD11b, CD86, CD206, CD209, and HLA-DR expression levels were similar in MDMs from P1 and healthy unrelated controls.

Article Snippet: Antibody , anti-CD206 , Miltenyi Biotec , #130-099-732 , Fluorochrome: PE.

Techniques: Fluorescence, Expressing

Journal: eLife

Article Title: IRF4 haploinsufficiency in a family with Whipple’s disease

doi: 10.7554/eLife.32340

Figure Lengend Snippet:

Article Snippet: Antibody , anti-CD206 , Miltenyi Biotec , #130-099-732 , Fluorochrome: PE.

Techniques: Plasmid Preparation, Isolation, Purification, Transfection, Construct, Generated, Recombinant, Sequencing, TA Cloning, Expressing, Mutagenesis, Staining, Migration, Software

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: International Journal of Molecular Sciences

Article Title: Neuropeptide Y Promotes Human M2 Macrophage Polarization and Enhances p62/SQSTM1-Dependent Autophagy and NRF2 Activation

doi: 10.3390/ijms232113009

Figure Lengend Snippet: Flow cytometric analysis of the M1 markers HLA-DR and CD16 and the M2 markers CD206 and CD163 on ( a ) M0, ( b ) polarized M(IFN-γ/LPS), and ( c ) polarized M(IL-10) macrophages. Macrophages (7 × 10 5 cells per mL) were stimulated or not with 10 −8 M NPY in the presence or absence of LPS in complete medium for 24 h and then analyzed for surface marker expressions by flow cytometry. Histograms show the percentages of positive cells (%) and the mean fluorescence intensity (MFI). Results are expressed as mean value ± SD of 4 independent experiments. Significance was determined by one-way ANOVA followed by Tukey’s post hoc analysis; *: p < 0.05, **: p < 0.01.

Article Snippet: To determine macrophage phenotypic surface markers, macrophages were stained with the following monoclonal antibodies (mAbs): phycoerythrin (PE)-CD163, fluorescein isothiocyanate (FITC)-CD206 and PE-Vio770-CD206 mAbs (Miltenyi Biotec), allophycocyanin (APC)-CD16 and APC-Alexa Fluor 750-HLA-DR (clone Immu357) mAbs (Beckman Coulter, Lane Cove, Australia), PE-CD1a and FITC-CD14 mAbs, or with isotype-matched control mAbs from PharMingen (PharMingen, San Diego, CA, USA) for 30 min at 4 °C.

Techniques: Marker, Flow Cytometry, Fluorescence