AHCYL1 senses SAH and mediates autophagy inhibition by SAH. (A) A schematic map of AHCY, AHCYL1 and AHCYL2. (B) Exogenous AHCYL1 readily binds with endogenous PIK3C3. Flag-AHCY, Flag-AHCYL1 and Flag-AHCYL2 were expressed in 293 T cells for Co-IP analysis. (C) Endogenous AHCYL1 interacts with endogenous PIK3C3 in U-2 OS, MEF and WI-38 Cells. (D) and (E) AHCYL1 is required for autophagy inhibition by SAH accumulation. In U-2 OS cells, KO-AHCYL1 increased LC3 puncta. ADOX decreased LC3 puncta and KO-AHCYL1 abolished the effect by ADOX. KD-AHCY showed similar results. (F) Demonstration of interactions between AHCYL1 and SAH, SAM, adenosine, Met, or Hcy by ITC. Bars and error bars represent mean values and SDs, respectively. The Two-way ANOVA was used for D and E. ns represents no significance, defined as P > 0.05. N = 5 in D and E.