Fig 2: RSK2 maintains genome stability in response to genotoxic stress.RSK2 is phosphorylated at both Ser227 and Thr577 in response to DNA damage. Activated RSK2 translocates to the nucleus and interacts with Atm, thus activating the Atm/p53-dependent DNA damage pathway, as well as p53-controlled cellular events. RSK2 KO relieves AD-induced cell cycle arrest at the G2/M check point and subsequently inhibited the DNA repair process, as indicated by the increased formation of ?H2AX foci.

Fig 3: miR-29a-3p and miR-30c-5p directly repress DNMT3A and rescue oxidative stress and premature senescence induced by DGCR8 knockdown. (A) Schematic illustration of miR-30c-5p (site 1) and miR-29a-3p (site 2) binding sites within the human DNMT3A 3' UTR (662–868) region, which was cloned downstream of the Renilla luciferase reporter gene in the psiCHECK-2 vector. Two predicted targeting sites and the corresponding mutations are listed. The mutagenised nucleotides are underlined and highlighted in red. The construct for the mutant 3' UTR contained both mutagenised sites (M1+M2). Data were normalised to firefly luciferase. (B) Luciferase reporter assay. 293T cells were co-transfected with luciferase reporters carrying either the wild-type DNMT3A 3' UTR (Dnmt3a) or the mutagenised DNMT3A 3' UTR (mDnmt3a), as well as 50 nM negative control mimic (miR-Control) or miR-29a-3p or/and miR-30c-5p mimics. Error bars indicate the standard error of the mean of three experiments. (**P < 0.01 and ***P < 0.001 compared to the miR-control). (C) The mRNA and protein expression levels of DNMT3A in DGCR8 knockdown hMSCs with or without miR-29a-3p or/and miR-30c-5p overexpression. The mRNA levels of DNMT3A were normalised to GAPDH. Error bars denote the standard error of the mean of three independent experiments (top). Immunoblot showing DNMT3A protein levels in DGCR8 knockdown cells with or without miR-29a-3p or/and miR-30c-5p forced expression (bottom). (D) Total ROS levels in DGCR8 knockdown hMSCs after miR-29a-3p or/and miR-30c-5p overexpression were measured using H2DCF-DA. Error bars denote the standard error of the mean of three independent experiments (**P < 0.01). Immunoblot showing the protein levels of SOD2 and Ras signalling components (bottom). (E) Mitochondrial membrane potential (MMP) in DGCR8 knockdown hMSCs after miR-29a-3p or/and miR-30c-5p overexpression was determined using JC-1. Representative fluorescence microscopy images in siGFP, siDGCR8, and mimic-transfected siDGCR8 hMSCs. Scale bar, 100 µm. (F) Western blot showing the protein levels of tumour suppressors and senescence markers, including p21, p53, and p-p53 (ser15), after miR-29a-3p or/and miR-30c-5p overexpression in DGCR8 knockdown hMSCs. (G) Mean growth rate of siGFP and siDGCR8 hMSCs co-transfected with miR-29a-3p or/and miR-30c-5p mimics, as determined by MTS. Error bars denote the standard error of the mean of three independent experiments (**P < 0.01).

Fig 4: Schematic describing a model for the role of Ser15 phosphorylation in p53-mediated transactivation. (1) p53 undergoes phosphorylation on Ser15. (2) This permits recruitment of histone acetyl-transferases (e.g. p300) (2) leading to acetylation of histones in the neighbouring vicinity (3) and subsequent relaxation of the chromatin (4). The inability to phosphorylate p53 at residue 15 (e.g. in the S15A mutant) impedes HAT recruitment and subsequent histone acetylation.

Fig 5: IBP is p53 dependently suppressed in response to DNA damaging agents. The expression of IBP was tested with Western analysis. (A) MCF-7 and ZR-75-1 which are human breast cancer cells with wild type p53 expression were treated with cisplatin for 24 h. (B) MCF-7 and ZR-75-1 cells were treated with doxorubicin for 8 h. (C) HCT116 p53+/+ cells and HCT116 p53-/- cells were treated with cisplatin for 24 h. (D) p53-RNAi MCF-7 cells were treated with cisplatin for different concentrations. Left was the RNAi effect of p53 in MCF-7 cells. GAPDH was used as a control.