Figure 8

FTO regulates CDK2 mRNA stability with YTHDF2 as the reader in an m⁶A-dependent manner.

(A, B) MeRIP-Seq data of HUVECs transduced with L-EV or L-FTO. Contents of m⁶A site in all types of RNAs (A) and distribution of total m⁶A peaks in distinct regions of mRNA transcripts (B) are presented. (C). Sequence of enriched motif displayed by HOMER. (D) Genes containing m⁶A-hypo peaks with altered protein expression are sorted out. (E) Abundance of m⁶A peak in the CDK2 transcript in HUVECs transduced with L-EV or L-FTO detected by MeRIP-Seq. Genomic location of its containing m⁶A site is annotated. (F, G) mRNA (F) and protein (G) expression of CDK2 in HUVECs exposed to NG or HG. β-actin is used as an internal control. n = 3 per group. (H, I) Cdk2 mRNA and protein levels detected in neural retinas of control and STZ mice. β-actin is used as an internal control. n = 3 per group. (J) MeRIP-qPCR analysis of m⁶A enrichment on CDK2 mRNA in HUVECs. n = 3 per group. (K) FTO-RIP-qPCR validates the binding between FTO protein and CDK2 mRNA in untreated HUVECs but not in HUVECs transduced with L-FTO^MU. n = 3 per group. (L). MeRIP-qPCR analysis of m⁶A enrichment on CDK2 mRNA in HUVECs transduced with L-EV, L-FTO^WT or L-FTO^MU. (M, N) mRNA (M) and protein (N) expression of CDK2 in HUVECs transduced with indicated lentivirus. GAPDH is used as an internal control. n = 3 per group. (O, P) CDK2 mRNA (O) and protein (P) levels in HUVECs transfected with scramble siRNA or FTO-siRNA. GAPDH was used as an internal control. n = 3 per group. (Q) CDK2 mRNA levels detected by qPCR in HUVECs transduced with indicated lentivirus at 0, 4 and 8 h post actinomycin D treatment. n = 3 per group. (R) YTHDF2-RIP-qPCR validation of YTHDF2 binding to CDK2 mRNA in HUVECs. n = 3 per group. (S) CDK2 mRNA levels detected by qPCR in HUVECs transfected with scramble siRNA or YTHDF2-siRNA at 0, 6 and 12 h post actinomycin D treatment. n = 3 per group. (T, U) CDK2 mRNA (T) and protein (U) levels in HUVECs transfected with scramble siRNA or YTHDF2-siRNA. GAPDH is used as an internal control. n = 3 per group. (V) CDK2 mRNA levels detected by qPCR in HUVECs transfected with scramble siRNA, FTO-siRNA, or FTO- and YTHDF2-siRNA. n = 3 per group. Data information: Data represent different numbers (n) of biological replicates. Data are shown as mean ± SEM. Two-tailed Student’s t test is used in (F–J, O–P, R–U). One-way ANOVA followed by Bonferroni’s test is used in (K–N, Q, V). NS: not significant (p > 0.05); *p < 0.05; **p < 0.01; and ***p < 0.001. Source data are available online for this figure.