Fig. 7: PAX2 regulates Kdm5a promoter activity in the context of folate deficiency. | Cell Death & Disease

Fig. 7: PAX2 regulates Kdm5a promoter activity in the context of folate deficiency.

From: Loss of KDM5A-mediated H3K4me3 demethylation promotes aberrant neural development by Wnt/β-catenin pathway activation

Fig. 7

A RT‒qPCR analysis of Tcf12, Pax2, and Lef1 mRNA expression in the spines and brains of E9.5 folate-deficient NTD model mice(n = 4) and control mice(n = 4). Gapdh was used as a loading control. *p < 0.05, **p < 0.01. B Analysis of KDM5A promoter-driven luciferase reporter activity in folate-deficient NE4C cells transfected with the control plasmid or the LEF1, PAX2, or TCF12 plasmid. The cells were harvested for the luciferase reporter assay after 48 h of transfection, n = 3, **p < 0.01. C PAX2 ChIP assays were performed using folate-deficient NE4C cells. The enrichment of PAX2 in the Kdm5a gene promoter was measured via RT‒qPCR. n = 3, **p < 0.01. D The PAX2 levels in folate-deficient NE4C cells was measured via Western blotting. GAPDH was used as a loading control. Protein levels were quantified using Image J software, n = 3. E RT‒qPCR analysis of Pax2 mRNA and Kdm5a mRNA expression in NE4C cells after siPax2 transfection for 48 h. GAPDH was used as a loading control. n = 3, **p < 0.01. F Immunostaining for PAX2 and KDM5A in NE4C cells transfected with siPax2 for 24 h. Images were acquired with a confocal microscope, and nuclei were stained with DAPI. n = 4, ***p < 0.001. G Immunostaining for KDM5A and PAX2 in the neural tubes of folate-deficient NTD model embryos at E13.5(n = 6) and control embryos(n = 6). Images were acquired with a confocal microscope, and the nuclei were stained with DAPI. H IGV visualization showing the differential H3K4me3 peaks of Pax2 promoters (2 kb upstream and downstream of the TSS) in NPSCs with folate deficiency or folate rescue. 10X FA: 40 mg/L synthetic folic acid supplementation. I H3K4me3 ChIP assays were performed on NPSCs with folate deficiency or folate rescue. Mouse IgG was used as a control. The enrichment of H3K4me3 in the promoters of the Pax2 was measured via RT‒qPCR. n = 3, p < 0.05, **p < 0.01, ***p < 0.001. J The PAX2 levels in NPSCs with folate deficiency or folate rescue was measured via Western blotting. GAPDH was used as a loading control. Protein levels were quantified using Image J software. All the above data are presented as the means ± SEM from three biological replicates.

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