Fig. 4: NR2E3’s agonist 11a specifically enhances NR2E3 to activate p53 in HeLa cells.

A 11a has a stronger inhibition of p53^+/+ HCT116 cells than p53^-/- HCT116 cells. The same number of p53^-/- and p53^+/+ isogenic HCT116 cells were seeded in 6-well plates and the indicated concentrations of 11a were achieved in each well. Photos of these cells were taken daily from day-0 to day-5. B shNR2E3 disrupted the 11a’s stimulation of the p53 reporter. 0.06 µg of the indicated shRNAs were co-transfected with the p53RE-SEAP reporter and SV40-RLuc into HeLa cells in 96-well plates. One day after transfection, 11a was added to achieve the indicated concentrations in each well. One day after 11a treatment, the luminescence was measured. The relative SEAP activity in the DMSO+scramble shRNA control was normalized to 1. p value was calculated by one-way Anova test and Bonferroni post-hoc test (shCon vs two shNR2E3). *p < 0.025; **p < 0.005; ***p < 0.0005. C 11a activated the p53 reporter much more in NR2E3 group than other groups. 0.01 µg of NR2E3 or its subfamily members were co-transfected with the p53RE-SEAP reporter and SV40-RLuc into HeLa cells in 96-well plates. One day after transfection, 11a was added to achieve the indicated concentrations in each well. One day after 11a treatment, the luminescence was measured. The relative SEAP activity in the DMSO control in mock control was normalized to 1. p value was calculated by one-way Anova test and Bonferroni post-hoc test (NR2E3 vs others). *p < 0.0125; **p < 0.0025; ***p < 0.00025. D HeLa cells were transfected with 1.2 µg FL or mock control in 6-cm dishes. 11a was added with fresh medium one day after transfection. Immunoblotting assays showed that one day treatment with 11a increased the acetylation of endogenous p53 at K319/386 in the control group (left). When HeLa cells were overexpressing the FL, immunoblotting assays showed the enhanced p53 acetylation at K319/386 and total protein level (right). One day treatment with 11a further increased the acetylation of p53 at K319/386. E HeLa cells were transfected with 3.8 µg shRNAs against NR2E3 or mock control in 6-cm dishes. 11a was added with fresh medium one day after transfection. Immunoblotting assays showed that knocking down NR2E3 prevented 11a from increasing the p53 acetylation at K319/386. F−J RNA sequencing was conducted in HeLa cells treated with 150 nM 11a or DMSO for one day. F Volcano plot of all transcripts of 11a and DMSO control. Red: growth inhibitory genes; Black: growth-promoting genes. G Changes of the selected genes were verified using qRT-PCR. p value was calculated by Student’s t-test with two tails. **p < 0.01; ***p < 0.001. H p53 pathway was enriched by 11a. I Metabolic glycolysis and oxidative phosphorylation pathways were enriched in DMSO controls. J Comparison of GSEA results between 11a vs DMSO and FL vs Control. Mean ± SD shown in all the histograms.