Fig. 3: NR2E3 R76W and R97H are loss of function mutations.
From: Orphan nuclear receptor NR2E3 is a new molecular vulnerability in solid tumors by activating p53
A Immunofluorescence analysis of co-localization of p53 and FL/R76W/R97H in HeLa cells two days after transfection with 0.02 µg of the indicated constructs in 8-chamber slides. Images were collected with confocal microscopy. Left: zoom-in images for the details. B R76W and R97H failed to enhance p300’s binding to p53 in co-immunoprecipitation assay with an anti-p300 antibody followed by immunoblotting analysis. 0.5 µg p53, 1.5 µg p300 and 1.5 µg FL/R76W/R97H were co-transfected into HeLa cells in 6-cm dishes for two days. C R76W and R97H failed to extend half-life time of p53 protein in HeLa cells two days after transfection with 1.2 µg of the indicated constructs in 6-cm dishes. The protein synthesis was ceased by 2 µg/mL of Cycloheximide and the cell lysates were collected at the indicated treatment time. D R76W and R97H failed to enhance HeLa cell nuclear extract’s binding to p53-DNA consensus in gel shift analysis. Cold (unlabeled) probe was loaded 50-fold more than the 32P-labeled probe. The indicated amount of plasmids were transfected into HeLa cells in 6-cm dishes for two days. E−L RNA-sequencing analysis of HeLa cells two days after transfection with 2.0 µg of the indicated constructs in 6-cm dishes. E Principal Component Analysis (PCA) plot of 9 samples. F Heat map of the most differentially expressed genes in FL, R97H and control groups. G Volcano plot of the transcripts of FL and R97H groups. Red: growth inhibitory genes. H The table summarized the number of differentially expressed genes in each comparison. I Expressions of growth inhibitory genes in (G) were verified using qRT-PCR. Mean ± SD shown. p value was calculated by Student’s t-test with two tails. **p < 0.01; ***p < 0.001. J Immunoblotting assay showed that R97H lost the ability to increase protein levels of p53, ATF3 and p21 in HeLa cells two days after transfection with 2.0 µg of the indicated constructs in 6-cm dishes. K−M GSEA studies of RNA-sequencing data. p53 (K), apoptosis and IFN-α (L) pathways were enriched in FL group. M Acetylation-related gene sets were enriched in FL group while Deacetylation-related pathway was enriched in R97H group. N R97H abolished the FL from acetylating p53 in the immunoblotting assay. HeLa cells were transfected with 0.6 µg p53, 0.2 µg GFP and 2.0 µg FL/R76W/R97H in 6-cm dishes for two days. GFP: transfection reference.
