cd31 biomarker  (AngioScore Inc)

Journal: Cell reports

Article Title: Targeting EP2 receptor with multifaceted mechanisms for high-risk neuroblastoma

doi: 10.1016/j.celrep.2022.111000

Figure Lengend Snippet: (A) Human 11q-deleted SK-N-AS cells with conditional KD of EP2 were generated using Tet-inducible lentiviral shRNA. EP2 shRNA was induced by doxycycline (0.5 μg/mL), and the efficacy of KD was validated by qPCR to measure EP2 mRNA levels. EP2 expression was significantly decreased in EP2 KD cells by >65% when compared with WT cells (n = 4; ***p < 0.001, t test). Data are presented as mean + SEM. (B) WT or EP2 KD SK-N-AS cells were inoculated (5 × 10 6 cells per site) into athymic nude mice (female, 6 weeks). After solid tumors were well established, animals were treated with doxycycline (50 mg/kg i.p.) daily to deplete EP2 in tumor cells. Tumor volumes were measured and compared (n = 10, F (1, 18) = 23.9, p = 0.0001; multiple comparisons: ***p < 0.001, two-way ANOVA and post hoc Dunnett’s multiple comparisons test). Data are presented as mean ± SEM. (C) Tumors formed by WT and EP2 KD cell lines were collected and displayed. (D) Xenografts formed by WT and EP2 KD cells were weighed and compared (n = 10; ***p < 0.001, t test). Data are presented as mean + SEM. (E) EP2 expression in WT and EP2 KD tumor tissues was examined by immunostaining (green fluorescence). Scale bar, 50 μm. (F) SK-N-AS cells were inoculated into athymic nude mice (female, 6 weeks) with two injection sites per animal: 5 × 10 6 cells and 10 × 10 6 cells on each flank side. After solid tumors were developed, vehicle or selective EP2 antagonist TG6-129 (10 or 20 mg/kg i.p.) was administered daily for 18 consecutive days. Tumor growth was monitored by measuring tumor volume daily. The SK-N-AS xenograft tumors formed by 5 × 10 6 cells and 10 × 10 6 cells did not differ in growth or size, so they were combined for comparisons between treatment groups (n = 8–10, F (2, 23) = 7.043, p = 0.004; multiple comparisons: p = 0.047 and 0.003 for 10 mg/kg treatment and 20 mg/kg treatment compared with control, respectively, two-way ANOVA and post hoc Dunnett’s multiple comparisons test). Data are presented as mean ± SEM. (G) Tumors were harvested after 18-day treatment for comparisons. All tumors were weighed and compared between treatment groups (n = 8–10, F (2, 23) = 8.645, p = 0.002; multiple comparisons: p = 0.003 for 10 mg/kg treatment and p = 0.005 for 20 mg/kg treatment compared with control, one-way ANOVA with post hoc Dunnett’s multiple comparisons test). Data are presented as mean + SEM. (H) Immunostaining for Ki-67 (green fluorescence) was performed to identify proliferating cells in subcutaneous tumor tissues. Ki-67 expression levels were measured via quantifying the fluorescence intensity using ImageJ software and compared among groups (n = 4–5, F (2, 10) = 39.87, p < 0.001; multiple comparisons: p = 0.004 for 10 mg/kg treatment and p < 0.001 for 20 mg/kg treatment compared with control, one-way ANOVA with post hoc Dunnett’s multiple comparisons test). Data are presented as mean + SEM. Scale bar, 50 μm. (I) Immunostaining for CD31 (PECAM-1, green fluorescence) was utilized to indicate the microvessel density in subcutaneous tumors. CD31 levels were assessed via quantifying the fluorescence intensity and compared (n = 4–5, F (2, 10) = 9.248, p = 0.005; multiple comparisons: p = 0.025 for 10 mg/kg treatment and p < 0.004 for 20 mg/kg treatment compared with control, one-way ANOVA with post hoc Dunnett’s multiple comparisons test). Note that nuclei within each tumor were counterstained with DAPI (blue fluorescence). Data are presented as mean + SEM. Scale bar, 50 μm.

Article Snippet: We next examined the expression of CD31 in NB from the four major human patient cohort studies on R2 database platform (SEQC, Kocak, Versteeg, and NRC) and found that the EP2 receptor consistently displayed positive correlation in expression with CD31 across all these patient datasets ( ).

Techniques: Generated, shRNA, Expressing, Immunostaining, Fluorescence, Injection, Control, Software

Journal: Cell reports

Article Title: Targeting EP2 receptor with multifaceted mechanisms for high-risk neuroblastoma

doi: 10.1016/j.celrep.2022.111000

Figure Lengend Snippet: (A) Mouse NXS2 NB cells (2 × 10 6 cells per site) were inoculated into immunocompetent A/J mice (female, 6 weeks). After solid tumors were detected, vehicle or TG6-129 (20 mg/kg i.p.) was administered to mice daily for 3 weeks. Tumor growth was monitored by measuring tumor volume daily and compared (n = 10, F (1, 18) = 8.257, p = 0.0101; multiple comparisons: ***p < 0.001, two-way ANOVA and post hoc Dunnett’s multiple comparisons test). Data are presented as mean ± SEM. (B) Tumors were harvested and weighed for comparison between treatment groups (n = 10; *p < 0.05, t test). Data are presented as mean + SEM. (C) Immunostaining for Ki-67 (green fluorescence) was performed to identify proliferating cells in tumors treated by vehicle or TG6-129 (n = 10; ***p < 0.001, t test). Immunostaining for CD31 (PECAM-1, green fluorescence) was utilized to indicate angiogenesis (n = 10; **p < 0.01, t test). Data are presented as mean + SEM. Scale bar, 50 μm. (D) Immunostaining for cytokines IL-1β and IL-6 (green fluorescence) was employed to indicate the inflammation in allograft tumors. Cytokine levels were assessed via quantifying the fluorescence intensity and compared (n = 5; ***p < 0.001, t test). Data are presented as mean ± SEM. Scale bar, 50 μm. (E) Immunostaining for c-Casp3 and c-PARP (red fluorescence) was performed to assess apoptosis in allograft tumors from mice treated by vehicle and TG6-129. Protein levels of c-Casp3 and c-PARP were measured via quantifying the fluorescence intensity and compared (n = 5; **p < 0.01 for c-Casp3 and ***p < 0.001 for c-PARP, t test). Note that nuclei within each tumor were counterstained with DAPI (blue fluorescence). Data are presented as mean ± SEM. Scale bar, 50 μm.

Article Snippet: We next examined the expression of CD31 in NB from the four major human patient cohort studies on R2 database platform (SEQC, Kocak, Versteeg, and NRC) and found that the EP2 receptor consistently displayed positive correlation in expression with CD31 across all these patient datasets ( ).

Techniques: Comparison, Immunostaining, Fluorescence

Journal: Cell reports

Article Title: Targeting EP2 receptor with multifaceted mechanisms for high-risk neuroblastoma

doi: 10.1016/j.celrep.2022.111000

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: We next examined the expression of CD31 in NB from the four major human patient cohort studies on R2 database platform (SEQC, Kocak, Versteeg, and NRC) and found that the EP2 receptor consistently displayed positive correlation in expression with CD31 across all these patient datasets ( ).

Techniques: Virus, Recombinant, Modification, SYBR Green Assay, CRISPR, Expressing, Plasmid Preparation, shRNA, Software, Fluorescence, Microscopy, Spectrophotometry

Journal: American journal of reproductive immunology (New York, N.Y. : 1989)

Article Title: Placental pericytes and cytomegalovirus infectivity: implications for HCMV placental pathology and congenital disease

doi: 10.1111/aji.12728

Figure Lengend Snippet: A. Time course analysis of HCMV-GFP infection of placenta villous cells. A graph indicating the number of infected HCMV-GFP positive villous fibroblasts (open bars), trophoblasts (gray bars), and placental pericytes (black bars) per 1.25×105 total cells over the time course 12, 24, 48 and 96 h post-infection. 5B. HCMV infected cultures of villous fibroblasts, trophoblasts, and placental pericytes at 96 h post-infection showing GFP positive cells in pericytes, villous fibroblasts, and trophoblasts 5C. (A) Phase contrast image of a tri-cell culture mixture of villous fibroblasts, trophoblasts, and pericytes representing the placental villous core. (B) A live/dead stain of the villous tri-cell culture. (C) Dual labeled IHC of the tri-cell villous culture. Villous trophoblasts stained with cytokeratin-7 antibody (brown), placental pericytes stained with CD31 antibody (red), and villous fibroblasts are unstained, color chart shown. All images have a total magnification of 200×.

Article Snippet: The placenta tri-cell culture was dual-labeled for antigenic biomarkers cytokeratin-7 (clone OV-TL 12/30, Millipore, Bedford MA), and CD31 (Millipore, Bedford MA) as previously described [ 18 , 19 ].

Techniques: Infection, Cell Culture, Staining, Labeling