Fig. 5: m6A regulates mRNA stability of FIP200.
a The mRNA levels of FIP200 after treatment with actinomycin D were measured by RT-qPCR to analyze the stability of FIP200 in NPCs cocultured with BMSCs. mRNA half-life (t1/2) was calculated, and statistical analysis was conducted with the Student t test (n = 3, ***P < 0.001, NPCs + CPR + BMSCs vs. NPCs + CPR). b FLAG and YTHDF2 levels in cells transfected with control and YTHDF2-FLAG plasmid were analyzed by Western blot. GAPDH was used as loading control. c YTHDF2 protein expression levels were normalized to GAPDH; the results are presented as mean ± SD. Statistical analysis was conducted by unpaired Student t test (n = 3, **P < 0.01, YTHDF2-FLAG vs. Vector). d The stability of FIP200 in YTHDF2-overexpressing NPCs cocultured with BMSCs was analyzed by RT-qPCR. mRNA half-life (t1/2) was calculated, and data are shown as mean ± SD. Statistical analysis was conducted with the Student t test (n = 3, ***P < 0.001, OE-YTHDF2 + NPCs + CPR + BMSCs vs. NPCs + CPR + BMSCs). e The interaction of FIP200-FLAG in NPCs transfected with control or YTHDF2-FLAG was analyzed by RIP-qPCR. FIP200-FLAG enrichment was normalized to input; the results are presented as mean ± SD. Statistical analysis was conducted by unpaired Student t test (n = 3, ***P < 0.001, Anti-Flag vs. Anti-IgG). f The interaction of FIP200 with YTHDF2 in control and ALKBH5 knockdown NPCs with or without BMSCs cocultured was analyzed by RNA IP. Data are shown as mean ± SD. Statistical analysis was conducted with one-way ANOVA with Dunnet’s multiple comparison test (n = 3, **P < 0.01, Co-NPCs/siALKBH5 + Co-NPCs vs. NPCs; **P < 0.01, siALKBH5 + Co-NPCs vs. Co-NPCs). g YTHDF2 and FIP200 protein levels in control, and YTHDF2-knockdown NPCs after BMSC coculture under compression for 36 h, as measured by Western blot. Protein expression levels were normalized to GAPDH; the results are presented as mean ± SD. Statistical analysis was conducted by unpaired Student t test (n = 3, **P < 0.01, siYTHDF2 vs. siControl). h FIP200 mRNA levels after treatment with actinomycin D were analyzed by RT-qPCR to examine the stability of FIP200 mRNA in ALKBH5 and YTHDF2-knockdown NPCs cocultured with BMSCs. Data are shown as mean ± SD. mRNA half-life (t1/2) was calculated, and statistical analysis was conducted with the Student t test (n = 3, #no significance, siALKBH5 + siYTHDF2 + NPCs vs. NPCs).
