Research Paper Volume 10, Issue 11 pp 3308—3326

Figure 1. ZNF185 is a transcription target of p53. (A) UCSC genome browser analysis showing layered H3K4me3 mark in different cell lines, CpG islands, DNase clusters, conservation in vertebrates, and TF binding within ZNF185 promoter. (B) Genomic locus of ZNF185 showing the promoter region with H3K4me1 and p53 ChIP-seq signals after MCF7 treatment with either DMSO or nutlin. (C) Identified p53 binding site (p53 bs) within ZNF185 promoter region and conservation analysis among primates. (D) Amplification of specific DNA fragments after ChIP performed in SaOs-2 Tet-On-p53-HA cells using HA antibody. (E) Luciferase activity assay in H1299 after transfection of pGL3-ZNF185 promoter and either empty vector, p53 WT, p53-R175H, or p53-R273H expression vectors. ** P<0.01, n=4. Western blot analysis of cell lysates confirms p53 overexpression. (F) Luciferase activity assay in H1299 after transfection of pGL3-ZNF185 promoter with either WT of mutated p53 bs and either empty vector or p53 WT expression vectors. ** P<0.01, n=3. Western blot analysis of cell lysates to confirm p53 overexpression. (G) RT-qPCR analysis of ZNF185 and CDKN1A mRNA levels in SaOs-2 Tet-On-p53-HA after induction of p53 expression with 2 µg/mL doxycycline. * P<0.05, ** P<0.01, n=3. Western blot shows p53 and p21 levels. (H) RT-qPCR analysis of ZNF185 and CDKN1A mRNA levels in H1299 after transfection with empty vector, p53 WT, p53 R175H, or p53 R273H expression vectors. ** P<0.01, n=3. Western blot shows p53 and p21 levels.