Journal: Cell cycle (Georgetown, Tex.)

Article Title: Exosomal circPVT1 derived from lung cancer promotes the progression of lung cancer by targeting miR-124-3p/EZH2 axis and regulating macrophage polarization.

doi: 10.1080/15384101.2021.2024997

Figure Lengend Snippet: Figure 2. LC-derived Exos induce macrophage polarization toward to M2 phenotype. Notes: After PMA induction, the morphology of THP-1 cells was observed under a microscope (A) (n = 3). qRT-PCR was applied to detect the expression of macrophage surface marker CD68 (B) and surface markers of M1 (iNOS and IL-1β) and M2 (CD206, CD163 and arginase-1) (D) (n = 3). The uptake of Exos in macrophages was inspected by fluorescence labeling of Exos (C) (n = 3). The protein levels of M2 markers CD206, CD163 and arginase-1 in macrophages were assessed by Western blot (E) (n = 3); **P < 0.01, ***P < 0.001, compared to the M group; LC, lung cancer; Exos, exosomes; PMA, phorbol-12-myristate-13-acetate; M, macrophage.

Article Snippet: The membranes were incubated with the primary antibodies against rabbit anti-human GAPDH (5174S, 1:1000, Cell Signaling, Boston, USA), CD9 (sc-13,118, 1:500, Santa Cruz, Texas, USA), CD81 (sc-166,029, 1:500, Santa Cruz, Texas, USA), TSG101 (sc7964, 1:500, Santa Cruz, Texas, USA), CD206 (sc-376,108, 1:500, Santa Cruz, Texas, USA), arginase-1 (sc-166,920, 1:500, Santa Cruz, Texas, USA), CD63 (ab134045, 1:1000, Abcam, MA, USA), CD163 (ab182422, 1:1000, Abcam, MA, USA) and EZH2 (ab186006, 1:2000, Abcam, MA, USA) at room temperature in a shaking table for 1 h. Following primary incubation, the membranes were washed with the washing solution for 3 × 10 min and then incubated with the secondary antibody against horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000, Beijing ComWin Biotech Co., Ltd., Beijing, China) for 1 h at room temperature.

Techniques: Derivative Assay, Microscopy, Quantitative RT-PCR, Expressing, Marker, Fluorescence, Labeling, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Exosomal circPVT1 derived from lung cancer promotes the progression of lung cancer by targeting miR-124-3p/EZH2 axis and regulating macrophage polarization.

doi: 10.1080/15384101.2021.2024997

Figure Lengend Snippet: Figure 5. Exosomal circPVT1 stimulates macrophage polarization to M2 type and enhances the biological function of LC cells. Notes: The A549 cells were transfected with oecircPVT1 or si-circPVT1. Exos were extracted from the transfected A549 cells (Exo- A-oecircPVT1 or Exo-A-si-circPVT1) and coincubated with macrophages (M+ Exo-A-oecircPVT1 or M+ Exo-A-si-circPVT1). Then, qRT- PCR was utilized to measure the mRNA level of circPVT1 in A549 cells (A) and Exo-A (B) as well as the expressions of CD206, CD163 and arginase-1 in M+ Exo-A-oecircPVT1 or M+ Exo-A-si-circPVT1 (C) (n = 3). The protein expressions of M2 markers in M+ Exo- A-oecircPVT1 or M+ Exo-A-si-circPVT1 were detected by Western blot (D) (n = 3). Subsequently, the proliferation, invasion and migration abilities of A549 cells that coincubated with M+ Exo-A-oecircPVT1 or M+ Exo-A-si-circPVT1 were determined by CCK-8 assay (E), Transwell (F) and cell scratch assay (G), respectively (n = 3). The mRNA expressions of M2 markers and miR-124-3p in the transfected macrophages were inspected by qRT-PCR (H) (n = 3); *P < 0.05, ***P < 0.001, compared to the Blank group; *P < 0.05, **P < 0.01, ***P < 0.001, compared to the M+ Exo-A-si-NC or M+ Exo-A-vector group; *P < 0.05, **P < 0.01, compared to the M/ Blank group; M, macrophage; LC, lung cancer; Exos, exosomes.

Article Snippet: The membranes were incubated with the primary antibodies against rabbit anti-human GAPDH (5174S, 1:1000, Cell Signaling, Boston, USA), CD9 (sc-13,118, 1:500, Santa Cruz, Texas, USA), CD81 (sc-166,029, 1:500, Santa Cruz, Texas, USA), TSG101 (sc7964, 1:500, Santa Cruz, Texas, USA), CD206 (sc-376,108, 1:500, Santa Cruz, Texas, USA), arginase-1 (sc-166,920, 1:500, Santa Cruz, Texas, USA), CD63 (ab134045, 1:1000, Abcam, MA, USA), CD163 (ab182422, 1:1000, Abcam, MA, USA) and EZH2 (ab186006, 1:2000, Abcam, MA, USA) at room temperature in a shaking table for 1 h. Following primary incubation, the membranes were washed with the washing solution for 3 × 10 min and then incubated with the secondary antibody against horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000, Beijing ComWin Biotech Co., Ltd., Beijing, China) for 1 h at room temperature.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Migration, CCK-8 Assay, Wound Healing Assay, Plasmid Preparation

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 Neuroinflammation

Article Title: Presence and activation of pro-inflammatory macrophages are associated with CRYAB expression in vitro and after peripheral nerve injury

doi: 10.1186/s12974-021-02108-z

Figure Lengend Snippet: Quantification of the number of pro-inflammatory and immunosuppressive macrophages in WT and Cryab −/− crushed sciatic nerves. Representative images and quantification of the number of CD16/32 + Iba1 + DAPI + ( a , c ) and CD206 + Iba1 + DAPI + ( b , d ) profiles in the sciatic nerves of naïve (N), and 3, 5, 7, 14, 21, and 28 days post-crushed WT (white bars) and Cryab −/− (black bars) animals; representative of 2 experiments, n =3–4 animals/group, bar = 20 μm, data represent mean ± sem, * p < 0.05 independent t -test. e Graph displaying the ratio of CD16/32 + cells to CD206 + cells for WT and Cryab − / − nerves

Article Snippet: The tissues were incubated overnight at 4 °C with rabbit anti-Iba1 (Wako Chemicals, 019-1974, 1:200) or goat anti-Iba1 (Novus Biologicals, NB100-1028, 1:100), rat anti-mouse CD16/32 (BD Biosciences, 553842, 1:500), goat anti-mouse CD206 (Santa Cruz Biotechnologies, sc-34577, 1:500), chicken anti-P0 (Abcam, ab39375, 1:500), rabbit anti-CD68 (Cell Signaling, 97778, 1:100), rabbit anti-arginase 1 (Cell Signaling, 93668, 1:50), rabbit anti-iNOS (BD Transduction Laboratories, 1:100; gift from Dr. Samuel David, McGill University), and DAPI (Invitrogen, D3571, 1:2000).

Techniques:

Journal: Materials Today Bio

Article Title: Toll-like receptor 9-overexpressing red blood cell biomimetic microbubbles adsorb cell-free DNA to relieve steatotic liver ischemia-reperfusion injury

doi: 10.1016/j.mtbio.2026.103558

Figure Lengend Snippet: RBC-MBs promotes the polarization of macrophages towards the M2 phenotype by inhibiting the TLR9-MyD88 and cGAS-STING pathways (A-F) RBC-MB treatment significantly attenuated CpG DNA-induced upregulation of pro-inflammatory cytokines (A-B), key proteins in the TLR9-MyD88 (C-D) and cGAS-STING (E-F) pathways. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h, followed by incubation with 0.5 mL RBC-MBs (concentration: 1 × 10 8 particles mL -1 ) for 24 h, and then subjected to western blot analysis (A, C, E). IL-1α, TNF-α, IL-6, TLR9, MyD88, STING, and cGAS levels were normalized to the β-actin level in each sample. p-NF-κB, p-TBK1, and p-IRF3 levels were normalized to their respective total protein levels (NF-κB, TBK1, and IRF3) in each sample. These normalized values were then used for statistical analysis (B, D, F), n = 6. (G) RBC-MB treatment significantly attenuated CpG DNA-induced upregulation of CD86 in macrophages. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h, followed by incubation with 0.5 mL RBC-MBs (concentration: 1 × 10 8 particles mL -1 ) for 24 h, then immunostained with an anti-CD86 antibody (green) and DAPI (blue, nuclei), and imaged by fluorescence microscopy. Representative images (left panel) and quantitative fluorescence graph (right panel) are shown. Scale bar: 50 μm, n = 6. (H-J) RBC-MB treatment significantly inhibited hepatic IRI-induced polarization of macrophages toward the M1 phenotype (H) and activation of the TLR9-MyD88 and cGAS-STING pathways (I-J). MASLD rats underwent 45 min of hepatic ischemia followed by reperfusion. At 0, 6, 12, 18, and 24 h post-reperfusion, the rats were treated with either Con-MBs or RBC-MBs. Primary macrophages were isolated from rat livers, then co-stained with CD68 and CD163 followed by flow cytometric analysis (H), n = 3/4, or subjected to western blotting (I-J). CD206, iNOS, TLR9, MyD88, STING, and cGAS levels were normalized to the β-actin level in each sample, and these normalized values were used for statistical analysis (J), n = 3. For (B, D, F, G right panel , H right panel , J), error bars represent mean ± standard deviation. P values were calculated using the unpaired Student's t-test (B, D, F, G right panel ) or one-way analysis of variance (ANOVA) (H right panel , J). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies used included: rabbit polyclonal antibodies against TLR9 (A14642, Abclonal), NF-κB (A2547, Abclonal), MyD88 (A0980, Abclonal), cGAS (A8335, Abclonal), IL-1α (A2170, Abclonal), TNF-α (A0277, Abclonal), IL-6 (A0286, Abclonal), CD206 (A8301, Abclonal), CD86 (A16805, Abclonal), CD47 (A1838, Abclonal), and ATP1A (A0643, Abclonal); rabbit monoclonal antibodies against STING (13647, CST, Beverly, MA, USA), p-NF-κB (3033, CST), p-TBK1 (5483, CST), TBK1 (ab40676, Abcam), p-IκB (2859, CST), IκB (4812, CST), p-IRF3 (4947, CST), and β-actin (AC026, Abclonal).

Techniques: Incubation, Concentration Assay, Western Blot, Fluorescence, Microscopy, Activation Assay, Isolation, Staining, Standard Deviation