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: 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