Fig. 6: Silencing of YTHDF2 partially restores FIP200 levels and rescues autophagy in ALKBH5-depleted NPCs cocultured with BMSCs.

a BCL2, BAX, and cleaved Caspase-3 protein levels in ALKBH5 or YTHDF2-knockdown NPCs under compression with or without BMSC coculture were determined by Western blot. GAPDH was used as a loading control. b, c The percentage of apoptotic ALKBH5 or YTHDF2-knockdown NPCs with or without BMSC coculture, as determined by FACS analysis. The sum of Annexin V⁺/PI⁺ cells (late apoptosis) and Annexin V⁺/PI^– (early apoptosis) cells was taken as the total apoptotic cells. The percentage of apoptotic cells as detected by FACS is presented as mean ± SD. Statistical analysis was conducted by one-way ANOVA with Dunnet’s multiple comparison test (n = 3, **P < 0.01, NPCs + BMSCs vs. NPCs; *P < 0.05, **P < 0.01, BMSCs/siALKBH5 + siYTHDF2 + BMSCs vs. siALKBH5 + BMSCs). d Expression levels of the autophagy-associated proteins FIP200, Atg7, Beclin1, P62, and LC3 in ALKBH5 or YTHDF2-knockdown NPCs under compression with or without BMSC coculture were determined by Western blot. GAPDH was used as a loading control. e TEM was applied to observe autophagic vesicles in ALKBH5 or YTHDF2-knockdown NPCs with or without coculture (scale bar: 2 μm [1700×]; scale bar: 1 μm [5000×]). f Expression levels of autophagy-associated proteins were normalized to GAPDH; the results are presented as mean ± SD. Statistical analysis was conducted with one-way ANOVA with Dunnet’s multiple comparison test (n = 3, *P < 0.05, **P < 0.01, NPCs + BMSCs vs. NPCs; *P < 0.05, **P < 0.01, NPCs + BMSCs/siALKBH5 + siYTHDF2 + NPCs + BMSCs vs. siALKBH5 + NPCs + BMSCs).