Fig. 1. Mutant p53 upregulates m⁶A reader YTHDF2 expression in LFS astrocytes and glioma cells.

a Immunostaining indicates iPSC-derived NPCs and astrocytes expressing their corresponding cell markers (SOX2 and NESTIN for NPCs, GFAP for astrocytes, OLIG2 for oligodendrocytes, and β-TUBULIN III for neurons). Scale bar, 50 µm. b m⁶A methylation dot blotting shows decreased m⁶A methylation in LFS astrocytes. Dot blotting is performed to identify polyadenylated mRNAs immunoblotted with anti-m⁶A antibodies (upper panel). Methylene blue staining of total mRNA is used as a loading control (lower panel). Dot density is measured by ImageJ. The blotting images represent the results of at least three independent experiments, while the bar charts depict technical replicates within a single experiment. c m⁶A methylation dot blotting indicates increased m⁶A methylation upon depletion of mutant p53 in LFS astrocytes. Dot blotting identifies polyadenylated mRNA isolated from shCtrl and shp53 transduced LFS astrocytes and immunoblotted with anti-m⁶A antibodies (upper panel). Methylene blue staining of total mRNA is used as a loading control (lower panel). Dot density is measured by ImageJ. The blotting images represent the results of at least three independent experiments, while the bar charts depict technical replicates within a single experiment. d Transcriptome analysis of the mRNA expression of known m⁶A regulators in WT and LFS astrocytes. Among 20 m⁶A regulators examined in this study, m⁶A reader YTHDF2 is significantly upregulated in LFS astrocytes compared with WT astrocytes (n = 2 biologically independent samples). e Immunoblotting indicates elevated YTHDF2 protein in multiple LFS and H1-p53(WT/G245D) astrocytes compared with WT and H1-WT astrocytes. f RT-qPCR analysis shows a decrease of YTHDF2 expression upon p53/mutant p53 knockdown in LFS astrocytes but not WT astrocytes (n = 3 biologically independent samples). g Depletion of p53/mutant p53 by p53 shRNAs leads to downregulated YTHDF2 protein expression in LFS astrocytes but not WT astrocytes. h RT-qPCR shows mutant p53s (p53(R175H) and p53(G245D)) upregulate YTHDF2 mRNA expression in WT astrocytes (n = 3 biologically independent samples). i Immunoblotting indicates upregulation of YTHDF2 protein following transduction of distinct mutant p53s (p53(R175H) and p53(G245D)) but not p53 into WT iPSC-derived astrocytes. The results are representative of at least three independent experiments (a–c, e, g, i). The data are presented as the mean ± SEM; two-way ANOVA with Bonferroni’s multiple comparison test (h, f); multiple t test (d). ***P < 0.001. ns not significant. Source data and exact P values are provided in the Source Data file.