Fig 1: NR4A1 is associated with LEF1 expression in PTC.A GSEA analysis of GSE27155 by Oncogenic Pathway database to identify the potentially enriched signatures in higher NR4A1 expression of PTC tissues. B The heatmap showed the RT-qPCR results of LEF1 target gene expression in NR4A1 knockout and control BCPAP and TPC-1 cells. Unpaired Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001. C Western blot analysis of LEF1 protein levels in NR4A1 knockout and control cells. D RT-qPCR results of LEF1 mRNA levels in NR4A1 knockout and control cells. The NR4A1 knockout group was compared to the control group, Student’s t test, ***P < 0.001. E Representative IHC stainings of NR4A1 (upper) and LEF1 (lower) proteins in PTC tumorous tissues with high or low NR4A1 expression. Scale Bars = 50 μm. F Statistical analysis was conducted based on the NR4A1, and LEF1 protein expression in the TMA with 42 cases of PTC. Chi-square test. G Western blot analysis to validate LEF1 knockout efficiency in BCPAP and TPC-1 cells. H Cell viability assay via CCK8 method to compare the proliferation rate between LEF1 depleted and control BCPAP and TPC-1 cells. One-way ANOVA test, **P < 0.01, ***P < 0.001. I Representative images and semiquantitative analyses of IHC staining of LEF1 protein in a TMA of paired PTC tumorous tissues and adjacent normal tissues. Chi-square test, **P < 0.01. Scale bars = 500 μm. J The heatmap illustrated the association of different clinical characters in PTC patients with high or low LEF1 expression. Statistical significance was assessed by the Chi-square test.
Fig 2: Akt phosphorylation was inhibited by Wnt antagonist during tail regeneration.a Protein content of LEF1, β-Catenin and p-Akt (Ser473, Thr308) in tail tissues from Wnt inhibition groups (IWP-4 and XAV939) were significantly reduced compared with that control (DMSO) group. b–e Relative level of proteins in Fig. 5a. Values represent mean ± SD, N = 3, nsp > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 3: NR4A1 crosstalk with epigenetic changes to transcriptionally increase LEF1.A Schematic diagram of the NR4A1-binding motif in the promoter of LEF1, predicted by the JASPAR database. B ChIP-PCR results of NR4A1 enrichment at the promoter of LEF1 in BCPAP and TPC-1 cells. C Schematic diagram of the luciferase reporter constructs contains the promoter of LEF1 with conserved wild-type (WT) NR4A1-binding motif and mutant strategy. D HEK293T cells were transfected with empty vector or NR4A1-overexpressed plasmids (left panel) to analyze the luciferase reporter activity driven by WT or mutant LEF1 promoter (right panel). Student’s t test, ***P < 0.001, ns, not significant. E Correlation between the methylation level of the probe cg06214925 (located near NR4A1-binding motif within LEF1 promoter) and the transcription level of LEF1 in PTC tumorous tissues in TCGA database. The linear correlation was determined by Pearson Correlation Coefficient. F Correlation between the expression levels of LEF1, NR4A1, histone acetyltransferase, and DNA demethylase in PTC tumorous tissues in TCGA database, performed by Pearson Correlation Coefficient analysis. G Western blot analysis of NR4A1 and LEF1 protein levels in BCPAP and TPC-1 cells transfected with empty vector or NR4A1-overexpressed plasmids. H RT-qPCR results of LEF1 mRNA levels in empty vector or NR4A1-overexpressed cells. Student’s t test, **P < 0.01, ***P < 0.001. I ChIP-qPCR analysis of H3K27ac and 5mC occupancy at the LEF1 promoter region around the NR4A1-binding site in TPC-1 cells. Student’s t test, **P < 0.01, ***P < 0.001. J Model depicting the role of NR4A1 in promoting the PTC cell proliferation via transcriptionally controlling LEF1.
Fig 4: Effects of kif3a knockdown on Wnt signaling in hDFCs and hDPCs. There was a decrease of Wnt signaling on (A and B) gene expression levels, revealed using reverse transcription-quantitative polymerase chain reaction analysis, and on (C) protein levels, revealed using Western blot analysis following 6 days osteoblastic induction in hDFCs and hDPCs. (D and E) mRNA and (F) protein expression levels of Wnt signaling molecules were markedly higher in the control cells, compared with the kif3a-knockdown cells following 100 ng/ml recombinant Wnt3a stimulation for 18 h. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.001, vs. control. hDFCs, human dental follicle cells; hDPCs, human dental pulp cells; CTR, control; kif3a KD, kif3a knockdown; Axin2, axis inhibition protein 2; Lef1, lymphoid enhancer-binding factor 1; p-GSK3β, phosphorylated glycogen synthase kinase 3β.
Supplier Page from Abcam for Anti-LEF1 antibody