Fig. 1: NR2E3’s full-length isoform instead of its short isoform activates p53 and suppresses cancer cells.

A The short failed to increase p53 protein level in HeLa cells two days after transfection with 1.2 µg of the indicated constructs in 6-cm dishes. Up: illustration of human NR2E3 proteins. DBD: DNA binding domain; LBD ligand binding domain, FL full-length isoform; and short: short-length isoform. Low: immunoblotting analysis of HA-tagged NR2E3 constructs and p53. B Identification of a nuclear localization signal at the C-terminus of NR2E3. Immunofluorescence was conducted with confocal microscopy and anti-HA antibody in HeLa cells two days after transfection with 0.02 µg of the indicated constructs in 8-chamber slides. C The short failed to activate the p53RE-FLuc reporter in HeLa cells two days after transfection with 0.01 µg of the indicated constructs in 96-well plates. SV40-RLuc was used as the transfection reference. The relative FLuc activity in the empty vector control was normalized to 1. p value was calculated by one-way Anova test and Bonferroni post-hoc test (each domain vs Con vs FL). *p < 0.0167; **p < 0.0033; ***p < 0.0003. D The short inhibited the FL’s activation of the p53RE-FLuc reporter in HeLa cells two days after transfection with both 0.01 µg FL and 0.01 µg or 0.03 µg of the indicated constructs in 96-well plates. SV40-RLuc was used as the transfection reference. The relative FLuc activity in the FL+empty vector control was normalized to 1. red *p values of FL+empty vector control vs low dose. p value was calculated by one-way Anova test and Bonferroni post-hoc test (FL vs low dose vs high dose). *p < 0.0167; **p < 0.0033; ***p < 0.0003. E The short inhibited the FL-induced HeLa cell apoptosis two days after transfection with both 1.2 µg FL and 2.8 µg of short or empty vector in 6-cm dishes in Annexin V-binding assay. p value was calculated by Student’s t-test with two tails. ***p < 0.001. F The FL activated the p53RE-FLuc reporter in six cancer cell lines two days after transfection with 0.01 µg FL in 96-well plates. These cells endogenously express wild-type p53, except H1299 cells with enforced p53 expression. The relative FLuc activity in mock control was normalized to 1. p value was calculated by Student’s t-test with two tails. **p < 0.01; ***p < 0.001. G The FL selectively rescued the wild-type transactivities of p53 mutations which partially reserved this transactivity in p53^-/- HCT116 cells. 0.01 µg of the indicated p53 constructs was co-transfected with the p53RE-FLuc reporter, SV40-RLuc and 0.01 µg FL/empty vector in 96-well plates. The relative FLuc activity in the p53^WT+empty vector control was normalized to 1. p value was calculated by Student’s t-test with two tails. ***p < 0.001. Left: Immunoblotting assay showed the protein levels of p53 mutations. GFP: transfection reference. H The FL enhanced the binding of acetylated p53 to p21 promoter in HeLa cells two days after transfection with 1.2 µg p53 plasmid and 2.4 µg FL in 10-cm dishes. Anti-acetylated p53 antibody was used in chromatin immunoprecipitation (ChIP) followed by qPCR analysis. BS: p53-binding site. I−M RNA-sequencing analysis of HeLa cells two days after transfection with 2 µg FL or empty vector. I Volcano plot of all transcripts. Red: growth inhibitory genes. J Heat map of the most differentially expressed genes. K Up-regulations of growth inhibitory genes were verified by qRT-PCR. p value was calculated by Student’s t-test with two tails. **p < 0.01; ***p < 0.001. L Gene sets of p53, apoptosis and HDAC were enriched in FL group. M Gene set of INFα was enriched in FL group. N RNA levels of growth inhibitory genes were measured by qRT-PCR in p53^-/- HCT116 cells two days after transfection with 4 µg FL in 6-cm dishes. p value was calculated by Student’s t-test with two tails. ^**p < 0.01; ***p < 0.001. Mean ± SD shown in all the histograms.