Fig. 3: ALKBH5 and the secondary m6A modification are involved in regulating NPC autophagy by BMSC coculture.
a The m6A/A ratio and m6A/AUCG ratio of total mRNA were determined by LC–MS/MS. Data are shown as mean ± SD. Statistical analysis was conducted by unpaired Student’s t test (n = 3, *P < 0.05, **P < 0.01; Co-NPCs + CPR vs. NPCs + CPR). b RNA dot blot was performed to analyze the m6A level of mRNA in NPCs with or without BMSC coculture. Methylene blue staining served as a loading control. c The relative m6A level in NPCs with or without BMSC coculture, as determined by colorimetric assay. Data are shown as mean ± SD. Statistical analysis was conducted by unpaired Student t test (n = 3, **P < 0.01, Co-NPCs + CPR vs. NPCs + CPR). d Relative mRNA levels of methyltransferases and demethylases were determined by RT-qPCR. The expression levels were normalized to the control group; the results are presented as mean ± SD. Statistical analysis was conducted by unpaired Student t test (n = 3, *P < 0.05, **P < 0.01, #no significance, NPCs + BMSCs + CPR vs. NPCs + CPR). e Protein levels of the methyltransferases METTL3, METTL14, and WTAP, and the demethylases FTO and ALKBH5, were measured by Western blot. Protein levels were normalized to GAPDH; the results are presented as mean ± SD. Statistical analysis was conducted by unpaired Student’s t test (n = 3, *P < 0.05, **P < 0.01, NPCs + BMSCs + CPR vs. NPCs + CPR). f The ALKBH5 knockdown efficiency by siALKBH5 was confirmed by Western blot. 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, siALKBH5 vs. siControl). g Protein levels of apoptosis- and autophagy-associated proteins after silencing of ALKBH5 with or without BMSC coculture, as determined by Western blot. h m6A/A ratio and m6A/AUCG ratio of the total mRNA in three groups, as determined by LC–MS/MS. Data are shown as mean ± SD. Statistical analysis was conducted by unpaired Student’s t test (n = 3, *P < 0.05, **P < 0.01, Co-NPCs + CPR vs. NPCs + CPR; **P < 0.01, Co-NPCs + CPR + siFIP200 vs. Co-NPCs + CPR). i The m6A level of mRNA in NPCs with or without BMSC coculture upon ALKBH5 knockdown was analyzed by RNA dot-blot analysis. Methylene blue staining served as a loading control. j Colorimetric assay to determine the relative m6A level in NPCs with or without BMSC coculture upon ALKBH5 knockdown. Data are shown as mean ± SD. Statistical analysis was conducted by unpaired Student t test (n = 3, *P < 0.05, Co-NPCs + CPR vs. NPCs + CPR; **P < 0.01, Co-NPCs + CPR + siFIP200 vs. Co-NPCs + CPR).
