Fig 2: 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).

Fig 3: Immunocytochemistry of C6 glioma cells. (A) Cells were stained with nestin antibody. Nuclei were counterstained with DAPI. Magnification, ×10. (B) Cells were stained with p53 antibody. Nuclei were counterstained with DAPI. Magnification, ×40. (C) Cells were stained with glial fibrillary acidic protein antibody. Nuclei were counterstained with DAPI. Magnification, ×40.

Fig 4: ZFP14 regulates p53 protein stability through MDM2. (A,D) The levels of p53 and actin mRNAs were measured in isogenic controls and ZFP14-KO HCT116 (clones #25 and #10) cells (A) or isogenic control and ZFP14-KO MCF7 (clone #4) cells (D). (B,E) The levels of p53, vinculin and actin proteins were measured in isogenic control and ZFP14-KO HCT116 (clone# 25) cells (B) or isogenic control and ZFP14-KO MCF7 (clone #4) cells (E) mock-treated or treated with cycloheximide (CHX) for indicated times. (C,F) The relative level of p53 protein in (B,E) was normalized to actin and the half-life of p53 protein was calculated by plotting the remaining protein over time. (G,H) pcDNA3-p53 and pcDNA3-2×-Flag-ubiquitin with or without pcDNA3-HA-ZFP14 were transfected in HCT116 cells followed by treatment with 20 µM MG132 for 5 h. Poly-ubiquitinated p53 levels in whole-cell lysates were measured by immunoprecipitation with mouse anti-p53 antibody followed by Western blotting with FLAG-HRP (G) or rabbit anti-p53 (H). (I,J) The levels of p53 and actin proteins were measured in isogenic control and ZFP14-KO HCT116 (clone# 25) cells (I) or isogenic control and ZFP14-KO MCF7 (clone #4) cells (J) mock-treated or treated with Nutlin for 16 h. (K) The levels of p53 and actin proteins were measured in isogenic control and ZFP14-KO HCT116 (clone# 25) cells mock-treated or treated with 250 µg/mL Dox for 18 h.

Fig 5: Immunocytochemical characteristic of glioma cells used in the experiment. Antibody staining against (?) nestin, (B) p53 and (C) C-X-C chemokine receptor type 4. Cytoplasms are stained with (A and C) Alexa 633 (pink) or (B) Alexa 488 (green). Nuclei are counterstained by DAPI (blue).