Fig. 5. p53 directly binds to the promoter of ITIH5.

A Schematic illustration of the pGL3-based reporter constructs used to detect the transcriptional activity of ITIH5 by luciferase assays. B, C Parts of the ITIH5 promoter, named P1, P2, P3 and P4, were transfected individually into 293 T cells with or without p53 overexpression. The expression levels of p53 were detected by western blotting (B). GAPDH was used as the loading control. The luciferase activity was measured (C). Data in C represent three independent experiments, **p < 0.01. D, E Schematic illustration of the key region of the ITIH5 promoter used for the pGL3-based reporter construct. Part of the ITIH5 promoter named P5 was transfected into ME4405 cells together with wild-type p53 or mutant p53. The luciferase activity was measured (D). The results represent three independent experiments, ***p < 0.001. The expression levels of p53 or mutants were detected by western blotting (E). GAPDH was used as the loading control. F, G P5 was transfected into Mel-RM (F) and Mel-CV (G) cells with or without p53 knockdown. The luciferase activity was measured. The results represent three independent experiments, ***p < 0.001. H Schematic illustration of the p53 wild-type binding site (BS, −770 to −755 bp) and the matching mutant (BSM) that were used in luciferase assays. I BS and BSM were transfected into HEK293T cells with or without p53 overexpression. The luciferase activity was measured. The results represent three independent experiments, ***p < 0.001. J BS and BSM were transfected into Mel-RM cells with or without p53 knockdown. The luciferase activity was measured. The results represent three independent experiments, ***p < 0.001. K, L ChIP analysis showed the binding of p53 to the promoter of ITIH5 in Mel-RM cells with or without p53 knockdown. An isotype-matched IgG was used as a negative control.