Fig 2: Expression of miR-211 and mRNA and protein expressions of SIRT1, Bcl-2, Bax, and p53 in lens tissues of mice(A) miR-211 expression and mRNA and protein expressions of SIRT1, Bcl-2, Bax, and p53 in mice lens; (B) strip chart of SIRT1, Bcl-2, Bax, and p53 proteins; (C) expressions of SIRT1, Bcl-2, Bax, and p53 proteins in mice lens; *, P<0.05 compared with the normal group.

Fig 3: ZFP14 is induced upon DNA damage and is a p53 target. (A–C) Correlation of ZFP14 expression with p53 expression in normal human tissues from GEPIA. (D) Schematic showing the various domains and motifs present in human and mouse ZFP14. AA; amino acids. (E–G) The levels of ZFP14, p21 and Actin mRNAs were measured by RT-PCR in MCF7 cells mock-treated or treated with 250 nM CPT (E) or RKO cells (F) and HCT116 cells (G) mock-treated or treated with 250 µg/mL Dox for 18 h. (H) The levels of Zfp14, p21 and Actin transcripts were measured in mouse C2C12 cells mock-treated or treated with 250 µg/mL Dox for 12-36 h. (I,J) qRT-PCR was used to measure the levels of ZFP14 and p21 mRNAs in MCF7 WT or p53-KO cells mock-treated or treated with 250 nM CPT (I) and RKO WT or p53-KO cells mock-treated or treated 350 µg/mL Dox for 18 h (J). (K) The levels of p53, ZFP14, p21 and Actin mRNAs were measured by RT-PCR in H1299 cells (H24-p53-15 clone) uninduced or induced to express p53 for 24 h. (L) Sequences of consensus p53-RE, the putative p53-REs located in ZFP14 intron 1 and p53-RE2 in the p21 promoter. Also shown are the locations of the p53-REs in the human ZFP14 and human p21 gene regions and the primer locations used for chromatin immunoprecipitation (ChIP) assays. (M) RKO cells were treated with 250 nM CPT for 16 h and the lysates obtained were used to perform ChIP assays using p53 antibody. PCR was performed using primers shown in (L) to determine the p53 binding to the ZFP14 promoter region. p53 binding to the p21 promoter upon DNA damage was used as a positive control.

Fig 4: Decrease in cancer cell growth and migration upon ZFP14 KO is p53-dependent. (A,B) Isogenic control and ZFP14-KO HCT116 (clone #10) cells: (A) cells or isogenic control and ZFP14-KO MCF7 (clone #4) cells (B) were plated for clonogenic assays and grown for two weeks, followed by fixation and crystal violet staining. ColonyArea plugin in ImageJ was used to quantify the relative colony areas, which are shown below each image. (C,D) Wound-healing assay was performed with isogenic control and ZFP14-KO HCT116 (clone #10) cells (C) or isogenic control and ZFP14-KO MCF-7 (clone #4) cells (D) for a period of 48 h. (E) The levels of p53, p21 and actin proteins were measured by Western blotting in isogenic control and ZFP14-KO HCT116 cells transfected with scramble, p53 siRNA#1 or p53 siRNA#2 for 72 h. (F) The cells treated as in (E) were plated for clonogenic assays 48 h after transfection and, after two weeks, the colonies were fixed and stained with crystal violet. (G) ColonyArea plugin in ImageJ was used to quantify the relative colony areas from (F) and plotted as percentage colony area. The area of isogenic control cells transfected with control siRNA was set at 100%. (H) The levels of p53, p21 and actin proteins were measured by Western blotting in isogenic control and ZFP14-KO MCF7 cells transfected with control, p53 siRNA#1 or p53 siRNA#2 for 72 h. (I) 72 h after transfection, the cells treated as in (H) were subjected to wound-healing assays for 48 h.

Fig 5: Human and mouse ZFP14 negatively regulate p53. (A–C) ZFP14, p53 and actin/GAPDH protein levels were measured by Western blotting in mouse C2C12 (A), human HCT116 (B) and human MCF7 (C) cells transiently transfected with either empty vector, mouse Zfp14-FLAG (A) or human HA-ZFP14 (B,C) for 24 h. (D–F) The levels of ZFP14 and actin mRNAs (left panels) or p53 and GAPDH/actin proteins (right panels) were measured in mouse C2C12 (D), human HCT116 (E) and human MCF7 (F) cells that were transfected with 75 nM scramble or ZFP14 siRNA for 72 h. (G,I) The levels of ZFP14 and actin mRNAs were measured in isogenic controls and ZFP14-KO HCT116 (clones #25 and #10) cells (G) or isogenic control and ZFP14-KO MCF7 (clone #4) cells (I). (H, J) The levels of p53 and actin proteins were measured in isogenic controls and ZFP14-KO HCT116 (clones #25 and #10) cells (H) or isogenic control and ZFP14-KO MCF7 (clone #4) cells (J).