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. 2023 Feb 16;24(4):e56325. doi: 10.15252/embr.202256325

Figure 1. Wild‐type p53 activates METTL14 expression in CRC cell lines.

Figure 1

  1. Schematic illustration of screening for transcription factors using ChIPBase (http://rna.sysu.edu.cn/chipbase3/index.php), PROMO alggen (http://alggen.lsi.upc.es/cgi‐bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3/) and TRAP (http://trap.molgen.mpg.de/).
  2. MEM (https://biit.cs.ut.ee/mem/) identified correlation between ELK1, p53 and METTL14.
  3. HCT116 and Lovo cells were transfected with empty vector and p53 plasmid. Forty‐eight hours after transfection, total RNA was then analyzed by qRT–PCR analysis. Data are presented as mean ± SD (biological replicates, n = 3; **P < 0.01, ***P < 0.001).
  4. HCT116, Lovo, and RKO cells were transfected with control or p53 siRNAs. Forty‐eight hours after transfection, total protein was then analyzed by western blot analysis.
  5. HCT116, Lovo, and RKO cells were transfected with empty vector and p53 plasmid. Forty‐eight hours after transfection, total protein was then analyzed by western blot analysis.
  6. Representative immunofluorescence staining of the p53 (green) and METTL14 (red) proteins in p53‐WT HCT116, Lovo, and RKO cells and p53‐MT HT29 (p53R273H), SW480 (p53R273H/P309S), SW620 (p53R273H), and SW1116 (p53A159D) cells. Nuclei were stained with DAPI (blue). Scale bars = 10 μm. The relative mean fluorescence density was analyzed by ImageJ.
  7. Correlation of METTL14 with p53 mRNA levels in p53‐WT and p53‐MT CRC cell lines from CCLE database (https://sites.broadinstitute.org/ccle). r is the Spearman's rank correlation coefficient.
  8. qRT–PCR analysis of METTL14 levels after transfection with empty vector, wild‐type, or mutant p53 plasmids in HCT116 (p53−/−) cells (biological replicates, n = 3; **P < 0.01, ns = no significance).
  9. Western blot analysis of METTL14 levels after transfection with empty vector, wild‐type, or mutant p53 plasmids in HCT116 (p53−/−) cells.
  10. Representative IHC images, and statistical analysis of immunoreactive score (IRS) of METTL14 expression in p53‐WT (n = 63) and p53‐MT (n = 41) CRC samples. The horizontal lines represent the median; the bottom and top of the boxes represent the 25 and 75% percentiles, respectively; and the vertical bars represent the range of the data. The insets show enlarged images of indicated p53‐WT and p53‐MT CRC tissues, respectively. Scale bars = 20 μm and 2 μm (inset).
  11. ChIP assay verified the potential p53‐binding site in the METTL14 promoter region in HCT116 and Lovo cell lines. Input fractions and IgG were used as controls.
  12. ChIP–qRT–PCR assay verified the potential p53‐binding site in the METTL14 promoter region in HCT116 and Lovo cell lines. IgG was used as a control. Data are presented as mean ± SD (biological replicates, n = 3; ***P < 0.001, ns = no significance).
  13. Luciferase activities of luciferase reporter plasmid containing wild‐type or mutant METTL14 promoter in control, wild‐type p53 or mutant p53‐overexpressing HCT116 (p53−/−) cells. pGL3 and Vector were used as controls. Data are presented as mean ± SD (biological replicates, n = 4; **P < 0.01, ns = no significance).

Data information: For (C, H, L, M), statistical significance was determined by the two‐tailed Student's t‐test. For (J), statistical significance was determined by the nonparametric Mann–Whitney test. β‐actin (ACTB) was used as a loading control, and p21WAF1/Cip1 was used as a positive control. The signal intensities were quantified by densitometry using ImageJ software and normalized to the intensity of the internal positive controls.

Source data are available online for this figure.