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: The tumor tissue sections were fixed by 4% PFA at room temperature for 15 min and permeabilized with 0.25% Triton X-100 at room temperature for 10 min. After blocking in 10% goat serum in PBS at room temperature for 1 h, the sections were incubated in primary antibodies at 4°C overnight: rabbit anti-Ki-67 (1:200, Biocare Medical, Cat# CRM 325B); rat anti-cluster of differentiation 31 (CD31) (1:200, eBioscience, Cat# 14-0311-82); rabbit anti-EP2 (1:100, Cayman Chemical, Cat# 101750); mouse anti-IL-1β (1:100, Cell Signaling Technology, Cat# 12242S); rabbit anti-IL-6 (1:100, Santa Cruz Biotechnology, Cat# sc-1265); rabbit anti-cleaved caspase-3 (1:200, Cell Signaling Technology, Cat# 9664T); rabbit anti-cleaved PARP (1:200, Cell Signaling Technology, Cat# 5625S).
Techniques: Generated, shRNA, Expressing, Immunostaining, Fluorescence, Injection, Software
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