Screening for TP53-inducible lncRNA candidates and verification of the binding of TP53 to these genes. (A) Venn plot showing the overlapping genes between lncRNAs with TP53-binding peaks from the ChIPBase v2.0 and TP53-regulated lncRNAs with translated ORFs. (B) The list of the candidates of TP53-inducible peptides. The number of supported samples for TP53 binding is shown in the list. (C) ChIP-seq tracks of normalized sequence tags show enrichment for TP53 along the TP53LC04 (also known as AC010501.1) loci in relative treatment visualized on IGV software. The box under the track represents the TP53 enrichment peak; “Dox+” represents samples treated with doxorubicin (dox), also called adriamycin (ADR); and “DDP+” represents the samples treated with cisplatin. (D) The putative TP53 binding motifs around the TSS of the candidates above. (E) ChIP-qPCR assays showing the binding of TP53 to the putative motifs above upon DNA damage. The HepG2 cells were treated with or without ADR for 24 h. BBC3 (also known as PUMA) was used as a positive control gene, and IgG was used as a negative control antibody. The values of ChIP efficiencies are given as the relative expression of TP53-IP normalized by IgG-IP. (F) Schematic diagram of the pGL4-minP vectors containing the wild-type or mutant putative TP53 binding motifs. The minimal TATA-box promoter is indicated as minP. (G) The luciferase reporter assays measuring the promoter activity of the indicated vectors in HepG2 treated or not treated with ADR for 24 h. The pGL4-minP vector was used as a negative control and a TP53 reporter vector as a positive control. (H) The indicated protein levels in HepG2 and HepG2TP53−/− cells with or without ADR treatment for 48 h. Data are represented as mean ± SEM. (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001.
