Furthermore, due to key structural and interactional differences, cholesterol exhibits a lower affinity for unsaturated lipid chains. The rigid, planar structure of cholesterol's steroid ring system is better suited to align with the straight, saturated acyl chains, creating a molecular shape mismatch with the kinked, unsaturated chains. However, the two methyl groups at the steroid also play an important role in cholesterol-lipid interactions by reducing sterol tilt in the bilayer, thereby allowing cholesterol to be oriented optimally ). Additionally, the kinks introduced by double bonds in unsaturated chains hinder tight packing with cholesterol, leading to weaker van der Waals interactions and further reducing affinity . Recent studies indicate that cholesterol is predominantly present in the outer leaflet of the plasma membrane, supporting the hypothesis of an asymmetric distribution ). However, this remains a subject of debate, as experimental methods to directly and conclusively address this question are still lacking. In our previous work, we demonstrated that altering cholesterol levels in the plasma membrane directly influences the unconventional secretory machinery of FGF2 by enhancing FGF2 recruitment at the inner plasma membrane leaflet and facilitating its translocation into the extracellular space, primarily through the modulation of PI(4,5)P 2 lipid properties .