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 cell science

Article Title: CREB regulates the expression of type 1 inositol 1,4,5-trisphosphate receptors.

doi: 10.1242/jcs.258875

Figure Lengend Snippet: Fig. 6. CREB regulates Ca2+ puffs produced by IP3R1. hR1 endo cells co-transfected with mCherry and pcDNA, VP16-CREB or KCREB plasmids (n=9 each) were loaded with Cal-520 and ci-IP3 followed by EGTA-AM, and imaged using a TIRF microscope. A UV-flash was delivered at 3 s to uncage ci-IP3. (A–C) Representative traces of Cal-520 fluorescence ratios (ΔF/F0) from the center of single Ca2+-puff sites (1.0×1.0 μm) obtained using hR1 endo cells co-transfected with mCherry and pcDNA (A), KCREB (B) or VP16-CREB (C). Boxed regions are shown enlarged on an expanded time scale at the top of each panel. (D) The number of Ca2+ puffs were significantly reduced in hR1 endo cells transfected with KCREB plasmid (red) compared with those in cells transfected with pcDNA (black). In contrast, the number of Ca2+ puffs were significantly increased in hR1 endo cells transfected with VP16-CREB (blue) plasmid compared with those in cells transfected with pcDNA. (E) The number of Ca2+-puff sites were also significantly diminished in hR1 endo cells transfected with KCREB plasmid compared with those in cells transfected with pcDNA. But the number of Ca2+-puff sites were slightly higher in hR1 endo cells transfected with VP16-CREB plasmid compared with those in cells transfected with pcDNA. (F) No difference was found in the amplitude of Ca2+ puffs following photolysis of ci-IP3 in hR1 endo cells transfected with pcDNA, KCREB or VP16-CREB. (G) The mean time of a 20%, 50%, 80%, or 100% rise (r) and decay (f) regarding the fluorescence of Ca2+ puffs evoked by photolysis of ci-IP3 did not differ between hR1 cells transfected with pcDNA, KCREB or VP16-CREB plasmid. Only mCherry-positive cells were considered for analysis. Data are presented as mean±s.e.m. Statistical significance was determined by Student’s t-test (unpaired, two-tailed). ***P<0.001, ****P<0.0001. ns, not significant.

Article Snippet: Phosphorylated IP3 receptor (Ser1756) antibody (#3760) was from Cell Signaling Technology and used at 1:1000 dilution.

Techniques: Produced, Transfection, Microscopy, Fluorescence, Plasmid Preparation, Two Tailed Test

Journal: Nature biotechnology

Article Title: Targeting herpes simplex virus with CRISPR-Cas9 cures herpetic stromal keratitis in mice.

doi: 10.1038/s41587-020-00781-8

Figure Lengend Snippet: Fig. 2 | HELP blocks HSV-1 infection of corneas and neurons in a prevention model. a, Flowchart for evaluating the antiviral effects of HELP in vivo. p24 HELP (100 ng), scrambled control mLP or 2 μl PBS (mock) was injected into the corneas of mice by intrastromal injection. After 24 h, the mice were infected with HSV-1 17syn+ (2 × 106 p.f.u. per eye). b, Deep sequencing analysis of on-target effects in HSV-1 and off-target effects in the mouse genome for UL8 gRNA; n = 4 mice. c, Deep-sequencing analysis of on-target effects in HSV-1 and off-target effects in the mouse genome for UL29 gRNA; n = 4 mice. d, Confocal imaging of HSV-1 and HELP in corneas. Mouse corneal sections were incubated with both anti-GFP (HELP) and anti-HSV-1 (VP5) antibodies. e, qPCR analysis of HSV-1 dissemination in the eye. f, p.f.u. analysis of HSV-1 dissemination in the eye. g, qPCR analysis of HSV-1 dissemination in the TG. h, P.f.u. analysis of HSV-1 dissemination in the TG. i, qPCR analysis of HSV-1 dissemination in the brain. j, P.f.u. analysis of HSV-1 dissemination in the brain. In e–j, the abundance of HSV-1 is shown as the number of viral genomes (VG) per diploid genome (DG); n = 4 mice; *P = 0.0286. k,l, Confocal analysis of HSV-1 in the whole brain (k) and TG (l). m, Confocal analysis of HELP in the TG after intracorneal injection. Data and error bars represent mean ± s.e.m.; unpaired two-tailed Mann–Whitney tests. The experiments were repeated twice with similar results.

Article Snippet: Slides were incubated with primary antibody against HSV-1 VP5 (1:200; Santa Cruz Biotechnology, sc56989) or rabbit primary antibody against GFP (1:1,000; GeneTex, 113617) in 1% BSA overnight at 4 °C.

Techniques: Infection, In Vivo, Control, Injection, Sequencing, Imaging, Incubation, Two Tailed Test, MANN-WHITNEY