Fig. 5. Three p53 regulators are affected by m⁶A methylation in HaCaT-T cells.

a, Protein relation analysis on m⁶A regulating proteins, p53, and genes with altered expressions in HaCaT cells after chronically exposed to low dose of NaAsO₂ (GSE97303) was performed by String. The genes in pink and blue rectangles differentially express in HaCaT cells chronically exposed to NaAsO₂ compared to HaCaT cells. The proteins in green rectangles are m⁶A regulating proteins. The genes in pink rectangles are involved in NaAsO₂-induced malignant transformation and associated with m⁶A methylation and p53. Straight lines represent two molecules are close related. b, The analysis of m⁶A sites in genes which are involved in NaAsO₂-induced malignant transformation and associated with m⁶A and p53 was performed in m⁶AVAR. The “green” means there are m⁶A sites in this gene from miCLIP/PA-m⁶A-seq experiments with high confidence. The “blue” indicates there are m⁶A sites in this gene from MeRIP-Seq experiments which is medium confidence. The “red” suggests there are m⁶A sites in this gene from transcriptome-wide predictions with low confidence. The area of “green”, “blue”, or “red” represents the relative counts of m⁶A sites in this gene. c-e, The mRNA levels of three genes including PRDM2 (c), MDM2 (d), and YY1 (e) were tested by RT-qPCR in HaCaT and HaCaT-T cells with introduction of two siMETTL3 (siMETTL3–1 and siMETTL3–2) and siControl. f, The representative images of PRDM2, MDM2, YY1, and GAPDH protein (left). Quantitative protein levels of PRDM2, MDM2, and YY1 protein in HaCaT and HaCaT-T cells with introduction of METTL3 siRNA were detected by western blotting (right). For c-f, n = 3 biological replicates, error bars indicate mean ± SEM. The P-values were determined by two-tailed t-test. “*” indicates a significant difference compared to control group (P < 0.05).