Journal: Advanced Science

Article Title: The Loss of an Orphan Nuclear Receptor NR2E3 Augments Wnt/β‐catenin Signaling via Epigenetic Dysregulation that Enhances Sp1‐β catenin‐p300 Interactions in Hepatocellular Carcinoma

doi: 10.1002/advs.202308539

Figure Lengend Snippet: Genetic ablation of NR2E3 accelerated liver tumor formation. A) Images of liver tumor from WT and Nr2e3 −/− mice injected with DEN (25 mg kg −1 ≈15 days old) at 24‐ and 46‐week time points (Male mice, N =6‐7 in each group and time point). Tumor nodule is indicated by red arrow (Top). A represents hepatocellular adenoma and C represent hepatocellular carcinoma in the H&E staining images (bottom). B) The number of tumor nodules per liver is shown at 24‐ and 46‐week time points. C) The ALT activity was measured. D) PCNA immunostaining images and the number of PCNA‐positive cells using liver tumor slides at 46‐week time point. E) A volcano plot of differential gene expressions using liver tumor RNA lysate from WT and Nr2e3 −/− KO at 24‐week time point ( N =3 in each group). Red dot or heatmap (right) indicates high expression of EGFR and EpCAM in the Nr2e3 −/− tumors. F) GSEA result exhibited the enrichment of Wnt/β catenin pathway in the Nr2e3 −/− KO liver tumors. G) Immunostaining sections of WT and Nr2e3 −/− liver tumor tissues with β catenin, EGFR, and EpCAM. Scale bar corresponds to 100 µm. All the data were analyzed by two‐tailed unpaired Student's t ‐test.

Article Snippet: The following antibodies either from Proteintech or Cell Signaling technology (CST) were used in immunoblotting or immunostaining analysis: p53 (Proteintech, Cat # 10442‐1‐AP), p21(Proteintech, Cat # 10355‐1‐AP), Bax (Proteintech, Cat # 50599‐2‐Ig), NR2E3 (Proteintech, 14246‐1‐AP), β‐catenin (CST, Cat # 8480) EGFR (CST, Cat # 4267), PCNA (Proteintech, Cat # 10205‐2‐AP), JAG1 (Proteintech, Cat # 66890‐1‐lg), PPARD (Proteintech, Cat # 60193‐1‐lg), Sp1(Proteintech, Cat # 21962‐1‐AP), non‐phospho β‐catenin (CST, Cat # 8814), and beta actin (Proteintech, Cat # 66009‐1‐lg).

Techniques: Injection, Staining, Activity Assay, Immunostaining, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: The Loss of an Orphan Nuclear Receptor NR2E3 Augments Wnt/β‐catenin Signaling via Epigenetic Dysregulation that Enhances Sp1‐β catenin‐p300 Interactions in Hepatocellular Carcinoma

doi: 10.1002/advs.202308539

Figure Lengend Snippet: Effects of NR2E3 depletion on liver cancer cell gene expression, phenotype, and features. A) Immunoblotting analysis was performed using HepG2 and Hep3B cell lysate derived from cells transduced with small hairpin RNA of scrambled control (CT), or two different small hairpin RNAs targeting NR2E3 (KO I & KO II) (Top). The TOP/FOP reporter luciferase activity was determined using CT, KO I, and KO II HepG2 and Hep3B cells (bottom). B) HepG2 and Hep3B cell growth was determined with or without NR2E3 depletion. C) Effects of sorafenib, an anticancer drug, HepG2 and Hep3B cell growth. D) Determination of cell migration potential of CT, KO I, and KO II HepG2 and Hep3B cells using a scratch assay. E) A Boyden chamber assay was performed using CT, KO I, and KO II HepG2 and Hep3B cells. Representative images are shown (Top) and invading cells per field were counted (Bottom). F) A self‐renewal capacity of HepG2 or Hep3B cells with or without NR2E3 depletion was determined by sphere formation assay. Images are shown (Top) and the number of spheres per field is presented as graphs (Bottom). G) Representative images of xenografted tumors stably transduced with scrambled control shRNA (CT) or shRNA targeting NR2E3 (KO II). (Top). Tumor volume is monitored after subcutaneous injection of the cells up to 27 days. H) H&E, PCNA, and β catenin staining images of CT vs. KO II xenografted tumors. I) Immunoblotting of PCNA, and β catenin using CT vs. KO II xenografted tumor tissues. Scale bar = 100 µm. Significance * ( p > 0.05) was determined by two‐tailed unpaired Student t ‐test.

Article Snippet: The following antibodies either from Proteintech or Cell Signaling technology (CST) were used in immunoblotting or immunostaining analysis: p53 (Proteintech, Cat # 10442‐1‐AP), p21(Proteintech, Cat # 10355‐1‐AP), Bax (Proteintech, Cat # 50599‐2‐Ig), NR2E3 (Proteintech, 14246‐1‐AP), β‐catenin (CST, Cat # 8480) EGFR (CST, Cat # 4267), PCNA (Proteintech, Cat # 10205‐2‐AP), JAG1 (Proteintech, Cat # 66890‐1‐lg), PPARD (Proteintech, Cat # 60193‐1‐lg), Sp1(Proteintech, Cat # 21962‐1‐AP), non‐phospho β‐catenin (CST, Cat # 8814), and beta actin (Proteintech, Cat # 66009‐1‐lg).

Techniques: Gene Expression, Western Blot, Derivative Assay, Transduction, Control, Luciferase, Activity Assay, Migration, Wound Healing Assay, Boyden Chamber Assay, Tube Formation Assay, Stable Transfection, shRNA, Injection, Staining, Two Tailed Test

Journal: Experimental and therapeutic medicine

Article Title: Study of dietary‑induced progression of psoriasis‑like mice based on gut macrophage polarization.

doi: 10.3892/etm.2023.11976

Figure Lengend Snippet: Figure 5. Protein expressions of Jagged1/Notch1/HES5 and TLR‑2/IκB‑α/NF‑κB p65 signaling pathway on skin lesions of imiquimod‑induced psoriasis model. (A and a) Western blot analysis of Notch signaling and NRP1 in skin lesions. The mammalian Notch signaling pathway consists of four Notch receptors (Notch 1, 2, 3, and 4) and five Notch ligands (Jagged 1, and 2, and Delta‑like 1, 3 and 4). HES5 is an important downstream target gene of Notch signaling pathway. NRP1 is involved in keratinocyte hyperproliferation of psoriasis accompanied by inflammation activated angiogenesis. (B and b) Western blot analysis of TLR‑2/IκB‑α/NF‑κB p65 signaling in skin lesions. Values are expressed as mean ± SD (*P<0.05, **P<0.01 vs. the normal control; #P<0.05, ##P<0.01, ###P<0.001 vs. the psoriasis group) and were statistically analyzed with one‑way ANOVA. NC, normal control; P, psoriasis; P + SF, psoriasis + SF; SF, stimulating food; p‑, phosphorylated.

Article Snippet: Proteins were transferred onto a PVDF membrane (MilliporeSigma), and then incubated with primary antibodies specific for NF‐κB p65 (cat. no. 6956), phospho‐NF‐κB p65 (cat. no. 3033), Toll‐like receptor‐2 (cat. no. 2229S), IKBα (cat. no. 9242), Jagged‐1 (cat. no. 70109s), Notch‐1 (cat. no. 3608s; all CST; 1:1,000), Hes‐5 (cat. no. ab25374; Abcam; 1:1,000) and anti‐β‐actin (cat. no. BM0627; Wuhan Boster Biological Technology, Ltd.; 1:2,000) overnight at 4 ̊C and then followed by incubation with horseradish peroxidase‐conjugated secondary anti‐mouse or anti‐rabbit IgG (cat. nos.

Techniques: Western Blot, Control