Fig. 5: Analysis of the biochemical and molecular changes observed in Pitx2KI mutant mice.
From: A missense mutation in Pitx2 leads to early-onset glaucoma via NRF2-YAP1 axis
A Expression of WT and mutant PITX2 proteins in 293T cells. The pCMV-Pitx2WT and pCMV-Pitx2R115L expression plasmids were transfected into 293T cells. Western blot analysis showed mutant PITX2 protein had the same abundance as WT. Antibody of Flag-tag was used to verify the specificity of PITX2 antibody. β-actin was used as the loading control. B Quantitative comparison of the WT and mutant PITX2 protein levels in transfected cells (n = 6 per cohort). C Subcellular localization of WT and mutant PITX2 proteins in COS7 cells. Immunocytochemistry study using anti-PITX2 antibody indicated that both WT and mutant PITX2 proteins were localized in the nucleus. D 293T cells were co-transfected with the indicated constructs. Flag immunoprecipitation, and blotting with anti-HA confirmed physical interaction. PITXWT-Flag was immunoprecipitated and the amount of co-precipitated NRF2-HA (left panels) or YAP1-HA (right panels) was determined by immunoblotting, shown are representative immunoblots. E PITX2R115L-Flag was immunoprecipitated and the amount of co-precipitated NRF2-HA (left panels) or YAP1-HA (right panels) was determined by immunoblotting, shown are representative immunoblots. F Quantification of immunoblots reveals the PITX2 p. R115L mutation decreases its interaction with NRF2 and YAP1 compared to WT (n = 4 in each group). G The transcriptional activity of WT and mutant PITX2 was detected with a luciferase reporter in transient transfections of 293T cells. All luciferase activities are normalized to β-galactosidase activity and shown as mean-fold activation compared with the vector. Five independent replicas were performed. The mean LEF-1 promoter luciferase activity with PITX2 expression was about 200,000 light units per 15 μg protein, and the β-galactosidase activity was about 65,000 light units per 15 μg protein. H RNA-seq results showing that several antioxidant-related genes are downregulated in mutant retinas. I RT-qPCR verified the RNA-seq results for the indicated genes (n = 12). *P < 0.05; **P < 0.01; ***P < 0.001. #, no significant difference. The data are represented as mean ± SEM.
